Transcript
Page 1: Handbook of Seed Physiology Applications to Agriculture
Page 2: Handbook of Seed Physiology Applications to Agriculture

Roberto L. Benech-ArnoldRodolfo A. SánchezEditors

Handbookof Seed Physiology

Applications to Agriculture

Pre-publicationREVIEWS,COMMENTARIES,EVALUATIONS . . .

“This is a very interesting and timelybook on seed physiology as it

applies to agriculture. The range of top-ics is broad, but they fit neatly underseveral general headings: (1) the rela-tionship between seeds and the soilin which they are planted, and strate-gies to improve seed performance inthe field; (2) behavior of seeds in thefield, emphasizing problems associatedwith dormancy, and lack of dormancy;(3) problems associated with seeds thatcan and cannot be stored in the drystate; and (4) the uses of commerciallyimportant seeds in an industrial con-text and the factors that influence theirquality.

Several of these topics have not beencomprehensively reviewed in recenttimes, making this an important andvaluable addition to the seed literature.There is much to be learned from thechapters, as might be anticipated giventhe quality and expertise of the authors.The reviews related to seed behavior areparticularly interesting since this areahas rarely been covered in other bookson seeds. Each chapter contains a verycomplete set of references, which is auseful guide to further reading. Many ofthe chapters will be extensively quotedfor years to come.”

J. Derek Bewley, PhDProfessor of Botany,University of Guelph, Canada

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More pre-publicationREVIEWS, COMMENTARIES, EVALUATIONS . . .

“Seeds are the beginning and theend of most agricultural prac-

tices. The ways in which seeds func-tion—their physiology, biochemistry,molecular biology, and genetics—arecritically important for agriculturalsuccess. But it is not only their use tohumankind that makes seeds impor-tant objects for study; their biologicalproperties, as agents for transmittingthe legacy of one generation to the next,have long stimulated the intellect andinvestigative zeal of scientists.

The editors have judiciously chosenareas that reflect all of seed biology andhave compiled an expert, authoritativeteam of seed scientists to write aboutthem. This text brings together an excit-ing collection of articles covering virtu-ally all of seed physiology important toagriculture, from seed germination, seedperformance, and seedling establishmentto dormancy, weed seeds, storage andlongevity, and quality of cereals andoilseeds. The information is up to date,complete, and comprehensive. The bookshould attract and satisfy agricultural-ists, seed scientists, and workers andstudents in related areas of biology. Iwarmly recommend this absorbing com-pendium for your study.”

Michael Black, PhDEmeritus Professor,King’s College, University of London, UK

“As the title indicates, Handbook ofSeed Physiology: Applications to Ag-

riculture updates several areas of seedbiology and physiology related to theagricultural and industrial use of seeds.The book is divided into four sectionsthat cover germination and crop estab-lishment, the effects of seed dormancyin crop production and quality, seedlongevity and conservation, and fac-tors associated with seed quality andindustrial uses of seeds.

This book covers a significant por-tion of current research related to thequality of seeds for both propagationand utilization. There is a good mix ofphysiological, genetic, biochemical, andmodeling approaches that are appliedto seed development, dormancy, ger-mination, and composition. The inte-gration of various levels of organiza-tion to understand how seeds behavein agricultural situations is an overalltheme of the book. The coverage inthese chapters offers enough detail forthe book to be used in graduate coursesin these topics, and also allows expertsto update their knowledge of the cur-rent status of related fields.”

Kent J. Bradford, PhDProfessor, Department of Vegetable Crops,Director, Seed Biotechnology Center,University of California, Davis

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More pre-publicationREVIEWS, COMMENTARIES, EVALUATIONS . . .

“This text is comprised of thirteenchapters on such topics as soil

physics and tillage, seedbed prepara-tion, grain quality for (human) foodand (animal) feed, crop emergence andestablishment, seed improvement, dor-mancy, and storage. This text is a valu-able and worthwhile contribution to theliterature on seed physiology. Indeed,the Handbook of Seed Physiology: Applica-tions to Agriculture delivers one’s expec-tations from the title.

The book’s value is in the breadth oftopics, authored by respective expertsin their fields, directed toward the agri-cultural applications of this knowledge,and brought together in one volume.This book is an excellent route intowhat one might term seed agronomyfor applied physiologists. It will be par-ticularly valuable for master’s coursesand other postgraduate teaching.”

Richard Ellis, BSc, PhDProfessor of Crop Physiology,Head of the School of Agriculture,Policy and Development,The University of Reading, Reading, UK

Food Products Press®The Haworth Reference Press

Imprints of The Haworth Press, Inc.New York • London • Oxford

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NOTES FOR PROFESSIONAL LIBRARIANSAND LIBRARY USERS

This is an original book title published by Food Products Press® andThe Haworth Reference Press, imprints of The Haworth Press, Inc.Unless otherwise noted in specific chapters with attribution, materialsin this book have not been previously published elsewhere in any for-mat or language.

CONSERVATION AND PRESERVATION NOTES

All books published by The Haworth Press, Inc. and its imprints areprinted on certified pH neutral, acid-free book grade paper. This pa-per meets the minimum requirements of American National Standardfor Information Sciences-Permanence of Paper for Printed Material,ANSI Z39.48-1984.

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Handbookof Seed Physiology

Applications to Agriculture

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FOOD PRODUCTS PRESSSeed Biology, Production, and Technology

Amarjit S. Basra, PhDSenior Editor

Heterosis and Hybrid Seed Production in Agronomic Cropsedited by Amarjit S. Basra

Seed Storage of Horticultural Crops by S. D. Doijode

Handbook of Seed Physiology: Applications to Agricultureedited by Roberto L. Benech-Arnold and Rodolfo A. Sánchez

New, Recent, and Forthcoming Titles of Related Interest:

Wheat: Ecology and Physiology of Yield Determinationedited by Emilio H. Satorre and Gustavo A. Slafer

Hybrid Seed Production in Vegetables: Rationale and Methodsin Selected Crops edited by Amarjit S. Basra

Encyclopedic Dictionary of Plant Breeding and Related Subjectsby Rolf H. J. Schlegel

Handbook of Processes and Modeling in the Soil-Plant Systemedited by D. K. Benbi and R. Nieder

Biodiversity and Pest Management in Agroecosystems, SecondEdition by Miguel A. Altieri and Clara I. Nichols

Molecular Genetics and Breeding of Forest Treesedited by Sandeep Kumar and Matthias Fladung

Concise Encyclopedia of Plant Pathology by P. Vidhyasekaran

Agrometeorology: Principles and Applications of Climate Studiesin Agriculture by Harpal S. Mavi and Graeme J. Tupper

Abiotic Stresses: Plant Resistance Through Breedingand Molecular Approaches edited by Muhammad Ashrafand Philip John Charles Harris

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Handbookof Seed Physiology

Applications to Agriculture

Roberto L. Benech-ArnoldRodolfo A. Sánchez

Editors

Food Products Press®The Haworth Reference Press

Imprints of The Haworth Press, Inc.New York • London • Oxford

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Published by

Food Products Press® and The Haworth Reference Press, imprints of The Haworth Press, Inc.,10 Alice Street, Binghamton, NY 13904-1580.

© 2004 by The Haworth Press, Inc. All rights reserved. No part of this work may be reproduced orutilized in any form or by any means, electronic or mechanical, including photocopying, microfilm,and recording, or by any information storage and retrieval system, without permission in writingfrom the publisher. Printed in the United States of America.

Cover design by Marylouise E. Doyle.

Library of Congress Cataloging-in-Publication Data

Handbook of seed physiology : applications to agriculture / Roberto L. Benech-Arnold, Rodolfo A.Sánchez, editors.

p. cm.Includes bibliographical references and index.ISBN 1-56022-928-4 (Case : alk. paper)—ISBN 1-56022-929-2 (Soft : alk. paper)1. Seeds—Physiology. 2. Seed technology. I. Benech-Arnold, Roberto L. II. Sánchez,

Rodolfo A.SB117.H27 2004631.5'21—dc22

2003021276

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CONTENTS

About the Editors xi

Contributors xiii

Preface xv

SECTION I: GERMINATION IN THE SOIL AND STANDESTABLISHMENT

Chapter 1. Seedbed Preparation—The Soil PhysicalEnvironment of Germinating Seeds 3

Amos Hadas

Introduction 3Environmental Requirements of Germinating Seed 5Soil Environment—Physical Aspects 9Seedbed Preparation, Characterization of Seedbed Attributes,

and Seedbed Environment Conditions and SeedGermination 20

Water Uptake by Seeds and Seedlings 24Seed-Soil Water Relationships 27Modeling Seed Germination and Seedbed Physical

Attributes 29Concluding Remarks 36

Chapter 2. The Use of Population-Based Threshold Modelsto Describe and Predict the Effects of SeedbedEnvironment on Germination and Seedling Emergenceof Crops 51

William E. Finch-Savage

Introduction 51Imbibition 54Germination 56Other Germination Models 72Postgermination Seedling Growth 72Threshold Models: Prediction of Germination and Emergence

Patterns in the Field 74

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Summary and Conclusions 83Appendix 84

Chapter 3. Seed and Agronomic Factors Associatedwith Germination Under Temperature and Water Stress 97

Mark A. Bennett

Introduction 97Seed Coats 98Seed Size 99Seed Water Uptake 101Radicle Emergence and Root System Development 104Genetic Links to Germination Temperature Limits 105Seed Production and Seed Vigor 106Sowing Depths and Planter Technology 108Tillage Systems and Soil Structure Effects 109Interactions with Seed Treatments and Other Crop

Protection Chemicals 110Screening Protocols for Germination Tolerance to Low

Temperature and Water Stress 112Concluding Remarks 114

Chapter 4. Methods to Improve Seed Performancein the Field 125

Peter Halmer

Introduction 125Changing Seed Form and Lot Composition 126Physiological Enhancement 132Physiological Responses to Enhancement 139Ecological Aspects of Seed Hydration 155Conclusions and Future Directions 156

SECTION II: DORMANCY AND THE BEHAVIOROF CROP AND WEED SEEDS

Chapter 5. Inception, Maintenance, and Terminationof Dormancy in Grain Crops: Physiology, Genetics,and Environmental Control 169

Roberto L. Benech-Arnold

Introduction 169Physiology of Dormancy in the Cereal Grain 170

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Physiology of Dormancy in the Sunflower Seed 177The Expression of Dormancy in Grain Crops 180Removing Dormancy at an Industrial Scale 182Genetics and Molecular Biology of Dormancy

in Grain Crops 183Environmental Control of Dormancy in Grain Crops 188Concluding Remarks 190

Chapter 6. Preharvest Sprouting of Cereals 199Gary M. PaulsenAndrew S. Auld

Introduction 199The Preharvest Sprouting Process 201Physiological Control of Preharvest Sprouting 204Quality of Products from Sprouted Cereals 206Measurement of Preharvest Sprouting 209Controlling Sprouting by Breeding 212Controlling Sprouting in the Field 214

Chapter 7. The Exit from Dormancy and the Inductionof Germination: Physiological and Molecular Aspects 221

Rodolfo A. SánchezR. Alejandra Mella

Introduction 221The Effects of Light Photoreceptors 222Embryo Growth Potential 224Endosperm Weakening 226Termination of Dormancy: Its Relationship

with the Synthesis and Signaling of Gibberellinsand ABA 233

Concluding Remarks 235

Chapter 8. Modeling Changes in Dormancy in Weed SoilSeed Banks: Implications for the Predictionof Weed Emergence 245

Diego BatllaBetina Claudia KrukRoberto L. Benech-Arnold

Introduction 245Dormancy: Definitions and Classification 246

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How Is Dormancy Level Expressed? 247Environmental Factors Affecting Dormancy Level of Seed

Populations 248Factors That Terminate Dormancy 250Conceptualizing the System with Modeling Purposes 252Modeling Dormancy Changes in Weed Seed Banks

As Affected by the Environment 253Concluding Remarks 264

SECTION III: SEED LONGEVITY AND STORAGE

Chapter 9. Orthodox Seed Deterioration and Its Repair 273Miller B. McDonald

Introduction 273The First Seed 273Seed Deterioration 275Mechanisms of Orthodox Seed Deterioration 280Free Radical Production 281Free Radicals and Their Effects on Lipids 282How Do Free Radicals Cause Lipid Peroxidation? 282What Is the Influence of Seed Moisture Content on Free

Radical Assault? 283Do Free Radicals Attack Only Lipids? 285Why Suspect Free Radical Attack on Mitochondria? 285How Are Seeds Protected Against Free Radical Attack? 288Raffinose Oligosaccharides and Their Protective Role 291Repair of Seed Damage 292Model of Seed Deterioration and Repair During Priming/

Hydration 295Conclusions 296

Chapter 10. Recalcitrant Seeds 305Patricia BerjakNorman W. Pammenter

Seed Characteristics—The Broad Picture 305Seed Behavior 306The Suite of Interacting Processes and Mechanisms

Involved in Desiccation Tolerance 312Drying Rate and Causes of Damage in Recalcitrant Seeds 317

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SECTION IV: INDUSTRIAL QUALITY OF SEEDS

Chapter 11. Processing Quality Requirements for Wheatand Other Cereal Grains 349

Colin W. WrigleyFerenc Bekes

Introduction 349The Range of Grain Species Used Industrially 349Cereal Grains and Our Diet 352Uses of Cereal Grains 357Wheat-Grain Quality Traits: A Molecular Basis 368Grain Hardness 380Starch Properties 380Conclusion 382

Chapter 12. Grain Quality in Oil Crops 389Leonardo VelascoBegoña Pérez-VichJosé M. Fernández-Martínez

Introduction 389Components of Grain Quality in Oil Crops and Factors

Influencing Them 391Oil Quality 392Meal Quality 406Breeding and Production Strategies 412

Chapter 13. The Malting Quality of Barley 429Roxana SavinValeria S. PassarellaJosé Luis Molina-Cano

Introduction 429Grain Structural Components That Affect Malting Quality 433Genotypic and Environmental Factors Affecting

Malting Quality 437Achieving Barley-Grain Quality Targets 443Conclusions 448

Index 457

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ABOUT THE EDITORS

Roberto L. Benech-Arnold, PhD, is Associate Professor of GrainCrops Production at the Department of Plant Production and Chair-person of the Plant Production Program of the School for GraduateStudies, both at the University of Buenos Aires, Argentina. He is theauthor or co-author of more than 100 professional papers, abstracts,and proceedings on various aspects of seed science. Dr. Benech-Arnold is Regional Representative for South America for the Interna-tional Seed Science Society (ISSS) and speaks internationally onseed physiology, particularly in relation to the physiology and molec-ular biology of dormancy in grain crops. In addition, he serves as In-dependent Research Scientist for the National Council for Scientificand Technical Research in Argentina at IFEVA—the AgriculturalPlant Physiology and Ecology Research Institute, and is also a mem-ber of many scientific and professional organizations.

Rodolfo A. Sánchez, PhD, is Professor of Plant Physiology at theFaculty of Agronomy and Chairperson of the Doctorate Program ofthe School for Graduate Studies, both at the University of BuenosAires, Argentina. He is the author or co-author of more than 150 pro-fessional papers, abstracts, and proceedings. Professor Sánchez is aGuggenheim Fellow and an internationally recognized scientist inseed physiology and photobiology. He serves as Superior ResearchScientist for the National Council for Scientific and Technical Re-search in Argentina. At present Professor Sánchez is the Director ofIFEVA—the Agricultural Plant Physiology and Ecology ResearchInstitute.

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ContributorsCONTRIBUTORS

Andrew S. Auld, Kansas State University.

Diego Batlla, is Research and Teaching Agronomist, Departamentode Producción Vegetal, Facultad de Agronomía, Universidad deBuenos Aires, Argentina; e-mail: <[email protected]>.

Ferenc Bekes, PhD, CSIRO Plant Industry, Canberra, Australia;e-mail: <[email protected]>.

Mark A. Bennett, PhD, is Professor, Department of Horticultureand Crop Science, The Ohio State University, Columbus, Ohio; e-mail:<bennett. [email protected]>.

Patricia Berjak, PhD, School of Life and Environmental Sciences,University of Natal, Durban, South Africa; e-mail: <[email protected]>.

José M. Fernández-Martínez, PhD, is Professor, Instituto de Ag-ricultura Sostenible (CSIC), Alameda del Obispo s/n, Córdoba, Spain;e-mail: <[email protected]>.

William E. Finch-Savage, PhD, Horticulture Research Interna-tional, Wellesbourne, Warwick, United Kingdom; e-mail: <[email protected]>.

Amos Hadas, PhD, Institute of Soil, Water and EnvironmentalSciences, ARO, the Volcani Center, Bet Dagan, Israel; e-mail:<[email protected]>

Peter Halmer, PhD, Germain’s UK, Hansa Road, King’s Lynn,Norfolk, United Kingdom; e-mail: <[email protected]>.

Betina Claudia Kruk, DrSci, is Research and Teaching Agrono-mist, Departamento de Producción Vegetal, Facultad de Agronomía,Universidad de Buenos Aires, Argentina; e-mail: <[email protected]>.

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Miller B. McDonald, PhD, is Professor, Seed Biology Program, De-partment of Horticulture and Crop Science, The Ohio State Univer-sity, Columbus, Ohio; e-mail: <[email protected]>.

R. Alejandra Mella, DrSci, is Research and Teaching Agronomist,Cátedra de Fisiología Vegetal, Facultad de Agronomía, Universidadde Buenos Aires, Argentina; e-mail: <[email protected]>.

José Luis Molina-Cano, PhD, is Scientist and Head of the CerealDivision, Centro Universitat de Lleida-Institut de Recerca i Tec-nologia Agroalimentaries (UdL-IRTA), Lleida, Spain; e-mail: <[email protected]>.

Norman W. Pammenter, PhD, School of Life and EnvironmentalSciences, University of Natal, Durban, South Africa; e-mail <[email protected]>.

Valeria S. Passarella, is Research and Teaching Agronomist, De-partamento de Producción Vegetal, Facultad de Agronomía, Uni-versidad de Buenos Aires, Argentina; e-mail <[email protected]>.

Gary M. Paulsen, PhD, is Professor, Kansas State University; e-mail:<[email protected]>.

Begoña Pérez-Vich, PhD, is Associate Scientist, Instituto de Agri-cultura Sostenible (CSIC), Alameda del Obispo s/n, Córdoba, Spain;e-mail: <[email protected]>.

Roxana Savin, PhD, is Adjunct Professor, Departamento de Pro-ducción Vegetal, Facultad de Agronomía, Universidad de BuenosAires, Argentina; e-mail: <[email protected]>.

Leonardo Velasco, PhD, is Associate Scientist, Instituto de Agri-cultura Sostenible (CSIC), Alameda del Obispo s/n, Córdoba, Spain;e-mail: <[email protected]>.

Colin W. Wrigley, PhD, Food Science Australia and Value-AddedWheat CRC, North Ryde (Sydney), New South Wales, Australia;e-mail: <[email protected]>.

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PrefacePreface

Seeds have always caught the attention of both plant physiologists andagriculturists. Plant physiologists have been attracted by the multiplicity ofprocesses that take place in such a small organ (i.e., desiccation tolerance,reserve deposition and utilization, dormancy, and germination); agricultur-ists, in turn, have been well aware from the beginning that the establishmentof the “next crop” and the quality of their end product depend largely on“seed performance.” Considerable progress has been made in recent de-cades in the field of seed physiology. The advancement made in some topicsof this discipline is now sufficient to suggest approaches toward solvingpractical problems. On the other hand, attempts to solve these problems of-ten raise issues or suggest approaches to more fundamental problems.

This book is a collection of chapters dealing with different aspects ofseed physiology, each one having strong implications in crop managementand utilization. The book has been divided in four major sections: (1) ger-mination in the soil and stand establishment; (2) dormancy and the behaviorof crops and weeds; (3) seed longevity and storage; and (4) industrial qual-ity of seeds. Each section is composed of chapters dealing with specific as-pects of an agricultural problem. Each chapter covers the most recent find-ings in the area, treated at a basic level (physiological, biochemical, andmolecular level), but depicting the way in which that basic knowledge canbe used for the development of tools leading to increase crop yield and/orimproved industrial uses of the grain.

Section I addresses different aspects of crop germination and establish-ment. The physics of the seed environment, together with seed behavior inthe soil in relation to seedbed preparation, are described in an introductorychapter of this section. The rest of the section is devoted to discussing seedresponses to temperature and water availability, modeling crop emergence,breeding for germination at low temperatures and water availability, andsuggesting techniques for improving crop germination performance in thefield.

Section II covers dormancy problems in crop production. The first twochapters consider problems derived from the lack of control we have on thetiming of exit from dormancy in grain crops: preharvest sprouting and thepersistence of dormancy until the next sowing or seed industrial utilization.In Chapter 7 the termination of dormancy and the induction of germination

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is analyzed at a physiological and molecular level, mainly on the basis ofthe knowledge accumulated for two model species: tomato and Daturaferox. The section is completed with a chapter on dormancy in weedy spe-cies and the possibility of considering it in the generation of predictivemodels of weed emergence.

Section III presents an update in the field of seed longevity and conserva-tion. The section is divided in two chapters dealing with orthodox and recal-citrant seeds, respectively.

Section IV considers aspects related to the industrial uses of seeds. Thesection has been divided in three chapters: one considering cereal grainquality for flour production, another dealing with industrial quality of oilcrops, and a third devoted to discussing the development of good maltingquality.

We attempted to give this book a different scope than other valuableworks published recently in the area of seed biology. For example, the bookSeed Biology and the Yield of Grain Crops, written by Dennis Egli (CABInternational, 1998), covers only limited aspects of seed biology related tocrop production (namely, those related to the determination of grain weight).On the other hand, the book Seeds: Physiology of Development and Germi-nation, written by J. Derek Bewley and Michael Black (Plenum Press,1994), is an excellent textbook on seed biology but is not focused on cropproduction. Similarly, the comprehensive Seed Development and Germina-tion, edited by Jaime Kigel and Gad Galili (Marcel Dekker, 1995), sets thestate of the art in seed science, without paying particular attention to the ap-plication of basic knowledge for the resolution of agricultural problems.The books Seeds: The Ecology of Regeneration in Plant Communities, ed-ited by Michael Fenner (CAB International, 1992), and Seeds: Ecology,Biogeography, and Evolution of Dormancy and Germination, written byCarol and Jerry Baskin (Academic Press, 1998), discuss aspects of seed bi-ology with the aim of understanding ecological processes. Seed Quality:Basic Mechanisms and Agricultural Implications, edited by Amarjit S.Basra (The Haworth Press, 1995) and Seed Technology and Its BiologicalBasis, edited by Michael Black and J. Derek Bewley (Sheffield AcademicPress, 2000) are most closely related to this work; however, our book ad-dresses aspects that are not covered in either Seed Quality or Seed Technol-ogy (i.e., dormancy of crops and weeds, models for predicting crop germi-nation in the field, etc.).

We would like to thank all the authors who have contributed to this pro-ject. We are also indebted to our editorial assistant Juan Loreti who carriedout very fine work. Our colleagues Antonio J. Hall and María E. Oteguiacted as reviewers for some of the chapters and made comments and sug-gestions that greatly improved them.

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SECTION I:GERMINATION IN THE SOIL

AND STAND ESTABLISHMENT

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Chapter 1

Seedbed Preparation—The Soil Environment of Germinating SeedsSeedbed Preparation—The Soil PhysicalEnvironment of Germinating Seeds

Amos Hadas

INTRODUCTION

Germination Processes in Seeds

Among the stages of the plant life cycle, seed germination and seedlingestablishment are the most vulnerable. The term germination includes se-quences of complex processes that lead to the initiation of growth in the qui-escent embryo in the seeds, seedling development, and emergence from thesoil. During seed germination, various stored substrates are reactivated, re-paired if damaged, and transformed into new building materials necessaryfor the initial growth of the embryo, its subsequent growth, and seedling es-tablishment in its natural habitat (Koller and Hadas, 1982). To initiate thearray of processes, the condensed, insoluble stored substrates must first behydrated and then hydrolyzed to their basic forms before they can be repro-cessed. The processes necessary to hydrate and reactivate enzymes, cellmembranes, and cell organelles require much more respiratory energy thanis required to maintain the dry seed (Bewley and Black, 1982).

The necessary sequential order of this complex array of processes, someof which may occur simultaneously and others in a serial, interdependentorder, must be maintained to ensure its culmination in measurable and irre-versible growth. To achieve this, the processes must be properly controlled,probably by endogenous growth regulators (Khan, 1975; Taylorson andHendricks, 1977). Many of the metabolic events that are known to occurduring germination may differ in their timing, both among the various or-gans of a particular seed and among seeds of different species (Mayer andPoljakoff-Mayber, 1989; Bewley and Black, 1982; Hegarty, 1978). More-over, the transitions from one activity to another must be triggered by eventsthat occur only when the appropriate thresholds, dictated and timed by en-dogenous regulators and/or varying environmental conditions, are reached.

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The latter include environmental factors such as water availability, aeration,temperature, nutrients, and allelopathy caused by external toxins, e.g.,allelochemicals (Currie, 1973; Come and Tissaoui, 1973; Koller and Hadas,1982; Bewley and Black, 1982; Martin, McCoy, and Dick, 1990; Corbineauand Come, 1995; Bradford, 1995; Kigel, 1995).

Environmental Conditions

Proper germination of seeds and seedling emergence and establishmentare critical processes in the survival and growth cycle of plant species ingeneral. This is especially true in agriculture, since these processes deter-mine uniformity, crop stand density, degree of weed infestation, and the ef-ficient use of the nutrients and water resources available to the crop and ulti-mately affect the yield and quality of the crop (Hadas, 1997; Hadas, Wolf,and Rawitz, 1985; Hadas et al.,1990). Seed germination and stand estab-lishment are especially critical under marginal environmental conditions.Under arid conditions (i.e., infrequent wetting, wide temperature fluctua-tions, and high evaporation rates), germinating seeds have to obtain theirwater from the rapidly diminishing soil water reserves and must overcomehardening soil seals formed at the soil surface. Many arid zone soils tend toslake upon wetting and then during the subsequent drying form hard cruststhat impose mechanical obstacles to seed emergence and stand establish-ment, cause improper aeration, or lead to high-temperature injuries. Espe-cially susceptible to these crusting conditions are minute seeds or seeds thatare close to the soil surface, where the decrease of soil water content and theincrease of soil seal resistance are fastest.

Where favorable ecological conditions prevail, other factors may decidethe success or failure of an agricultural crop. Among these are seed devel-opment processes on the parent plants (Fenner, 1991; Gutterman, 1992),soil temperature (Probert, 1992), sensitivity to light (Scopel, Ballare, andSanchez, 1991), seed burial, and depth regulation during dispersion andwetting (Koller and Hadas, 1982). Overgrazing, compaction caused by ve-hicular and animal traffic, irregular spatial dispersion and placement depthof seeds, and inadequate seedbed preparation are among adverse environ-mental factors. Obviously, knowledge of the specific physiological require-ments of the various species of seeds and their physical interrelations withtheir environment, including climatic conditions, are of the utmost impor-tance in ensuring successful seed germination and stand establishment.

This chapter is devoted to analyzing the soil physical environment ofgerminating seeds with the final aim of establishing the basis for optimiza-tion of seedbed preparation. To achieve this aim, the chapter has been struc-

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tured in the following way: the first and second sections briefly analyze theenvironmental requirements for seed germination (i.e., water, temperature,aereation, and soil mechanical aspects) and the physics of the soil environ-ment, respectively; the third section gives a characterization of seedbed at-tributes; the fourth section briefly discusses the biophysics of water uptakeby seeds and seedlings; and the fifth section describes the physics of watermovement from the soil matrix toward the germinating seed. Finally, and onthe basis of all elements described in previous sections, the possibility ofmodeling seedbed attributes for optimization of stand establishment is dis-cussed in the sixth section.

ENVIRONMENTAL REQUIREMENTSOF GERMINATING SEED

Seeds are self-contained units, in contrast to the plants that develop aftergermination, due to the materials stored in the seeds. Environmental re-quirements for germination are fewer and simpler than those for whole-plant development, so germination is relatively independent of the environ-ment for a considerable period of seedling development. This assumption isbased on the observation that a seedling does not photosynthesize; there-fore, it requires neither light (except for regulatory or triggering functions)nor CO2 for its proper development until the seedling breaks through thesoil surface. Nevertheless, other environmental factors are needed, such aswater, temperature, and oxygen.

Water Requirements

The effects of soil water on germinating seeds are difficult to define in bi-ological terms, since soil water content and soil water potential are interde-pendent with soil constituents, their concentrations, and the scale and direc-tion (draining or wetting) of the processes (Collis-George and Lloyd, 1979;Marshal et al., 1996). Water uptake by seeds is a prerequisite for proper ger-mination, and under normal conditions, water uptake from the moist soildepends on the properties to water of the seed and the soil (Hegarty, 1978;Koller and Hadas, 1982).

The amount of water required by a seed for germination itself is verysmall. Water flow from the soil into the seed is driven by the water potentialdifferences between the seed and the soil and is controlled by the soil con-ductivity to water. The total water potential of a dry seed, seed, is very lowcompared to that of the soil, and the seed can draw water rapidly from thesoil it comes in contact with. The driving force, water potential gradient, de-

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fined as ( = seed – soil)/distance, is very large at the onset of imbibi-tion and decreases as the imbibing seed reaches the required hydrationlevel. Whether the amount of water taken up will suffice for germination de-pends on the water energy status of the seed and the adjacent soil total waterpotential, soil (Koller and Hadas, 1982; Hadas, 1982; Bradford, 1995).Greater amounts of water are required for seedling development in the laterpart of the seedling growth than during the hydration stages because of therequirements of the radicles and root hairs (Hadas and Stibbe, 1973).

Species and cultivars may differ markedly in their water requirements forgermination, and these differences have been attributed to the various en-demic soil water regimes to which they were adapted (Bewley and Black,1982; Koller and Hadas, 1982) and the differing soil physical conditions en-countered during germination.

Temperature Requirements

Temperature affects both the soil properties with respect to water and thebiological activity of seeds. Soil temperature varies greatly, both diurnallyand seasonally, and is dependent on soil moisture, structure, layering, andsoil color, as well as the site aspect and latitude (Marshal et al., 1996; vanWijk, 1963). The various effects of temperature on the rate of germinationand the total germination have been discussed extensively (Mayer andPoljakoff-Mayber, 1989, Chapter 2). For germination to occur, the tempera-ture of the seed environment should fall within a favorable, species-specificrange.

Cardinal temperatures for germination are the base, maximum, and opti-mal temperatures, which are, respectively, the temperature below or abovewhich no germination will occur and at which the faster germination rate isobserved (see Chapter 2 in this book). Favorable temperature ranges, spe-cific germination-enhancing conditions of diurnal or seasonal thermal peri-odicity, induction of secondary dormancy, and the combined effects of wa-ter stress and temperature vary among species (Kigel, 1995; Hegley, 1995;Benech-Arnold and Sanchez, 1995). Germination is greatly affected by theinteractions between temperature, water potential, and water flow in the soiland by variations in the Q10 factors of the effective seed biological activityrates (Allrup, 1958; Bewley and Black, 1982; Meyer and Poljakoff-Mayber,1989). The adverse effects of moisture stress on germinating seeds intensifyas temperatures rise (McGinnies, 1960; Evans and Strickler, 1961) and maypersist beyond the germination stages and extend into emergence and seed-ling growth stages through their strong effects on radicles and rootletgrowth.

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Aeration (Oxygen and CO2 ) Requirements

The aeration regime (i.e., rates of gaseous exchange) greatly affects soilbiological activity and the competition for oxygen with germinating seeds.However, their effects are complex and are difficult to define in biologicalterms (Currie, 1973; Collis-George and Lloyd, 1979). Such a definition re-quires knowledge of the interrelationships between complex diffusion pro-cesses (in air-filled pores in water films) that control oxygen supply and dis-sipation of respiratory and decomposition by-products (CO2, N2, NO2, H2S,ethylene, methane). Oxygen is required in germination as a terminal elec-tron receptor in respiration and other oxidative processes of a regulatory na-ture (Roberts and Smith, 1977). Low oxygen availability reduces or evenprevents germination in most species (Morinaga, 1926; Bewley and Black,1982; Corbineau and Come, 1995). Oxygen supply to support the metabolicactivity becomes decisive at a very early stage in germination, and oxygen-requiring metabolic activity is detected at an early stage of germination, in-dicated by a sharp rise in the respiration rate of seeds (Meyer and Poljakoff-Mayber, 1989). Another rise in respiration marks the beginning of thegrowth stage and radicle emergence. In between is a short period of con-stant respiration rate and oxygen consumption. Very often a conflict devel-ops between oxygen supply and water supply to germinating seeds, whicharises from the very low solubility and diffusivity of oxygen in water. Oxy-gen supply is greatly affected by the thickness of the water film covering thegerminating seed and the hydrated seed coat (Come and Tissaoui, 1973), es-pecially in seeds that have a swollen mucilaginous cover with very lowdiffusivity to oxygen (Heydecker and Orphanos, 1968; Witztum, Gutter-man, and Evenari, 1969). Nevertheless, a few species, such as aquaticplants, are able to germinate under reduced oxygen or even anoxic condi-tions (Rumplo et al., 1984; Taylor, 1942). Seeds rich in fatty or starchy stor-age substances stop germinating when the oxygen level falls below 2 percentand lower, respectively (Al-Ani et al., 1982, 1985). Oxygen requirementsincrease with soil temperature and under light and/or water stress (Smokeet al., 1993; Gutterman, 1992).

Low CO2 concentrations have been found to stimulate germination butmay at times affect it in combination with ethylene (Corbineau and Come,1995). Oxygen requirement and effects of oxygen and CO2 concentrationson germination are rather complex and may be not fully understood (Bewleyand Black, 1982; Meyer and Poljakoff-Mayber, 1989; Come and Cor-bineau, 1992).

Good aeration and gaseous exchange attained in well-structured, aggre-gated soil beds greatly assist germinating seeds, since the CO2 and ethylene

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produced can easily diffuse out of the soil so that seed dormancy and germi-nation retardation in CO2-sensitive species are relieved (Bewley and Black,1982; Corbineau and Come, 1995). The depth distributions of oxygen,CO2, and ethylene concentration depend on soil temperature, soil air-filledporosity, and the exchange, consumption, and production of these gases(Smith and Dowdell, 1974; Marshall, Holmes, and Rose, 1996). Soil crust-ing and compaction may have deleterious effects on gas exchange and, inturn, on seed germination (Richard and Guerif, 1988a,b).

Soil Mechanical Impedance

Soil is a porous material made of particles of varied sizes and origins thatform a matrix which exhibits a degree of resilience under mechanical stress,described as mechanical strength. Soil strength is a compound manifesta-tion of soil mechanical properties (cohesion, angle of internal shear, com-pressibility) and depends on soil density, constituents, water content, andsoil structure (Gill and van den Berg, 1967; Marshall, Holmes, and Rose,1996). It increases with increasing bulk density (soil slaking, shrinking, andcompaction) and decreases with increasing water content. Soils low in or-ganic material or high in silt fractions tend to deform plastically and to com-press easily, and to form seals under the impact and slaking action of rain-drops or under instant flooding by water or irrigation (Marshall, Holmes, andRose, 1996). Soil seals—thin, dense soil crusts—impede the germinationand emergence of seedlings by restricting gaseous exchange and infiltrationof water and by imposing a mechanical obstruction to emerging seedlings,or by any combination of these effects. Germination and final emergenceare reduced as the seal strength increases and/or as the moisture content de-creases (Arndt, 1965a,b; Richards, 1965; Hanks and Thorp, 1957; Hadasand Stibbe, 1977).

Adverse effects on seed germination and seedling development similarto those of seals are caused by soil compaction (Hadas, 1997; Hadas,Larson, and Allmaras, 1988; Hadas, Wolf, and Rawitz, 1983, 1985; Hadaset al., 1990). Increased soil strength is reflected not only in soil resistance toroot proliferation, seed swelling, or tuber expansion, but also in restrictedseedling emergence due to soil seals (Marshall, Holmes, and Rose, 1996).Nevertheless, under arid or semiarid situations, where soil moisture condi-tions are marginal, some soil compaction over the sown seeds has beenfound to improve germination and emergence (Hudspeth and Taylor, 1961;Dasberg, Hillel, and Arnon, 1966) and has been adopted as a common agro-nomic practice.

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SOIL ENVIRONMENT—PHYSICAL ASPECTS

Soil is seldom an ideal environment, and it can be quite hostile to germi-nating seeds and emerging seedlings. Yet soils form the natural habitat inwhich most seeds germinate and the environment with which they interactand with which they establish themselves successfully, provided that thesoil system and its constituents meet their requirements.

The Soil—A Three-Phased System

Soil is a three-phased system comprising solids (predominantly miner-als, e.g., weathered primary parent materials, secondary particles—mainlyclays—and organic matter), liquid (water and dissolved salts), and gases(a mixture in varying proportions).

The Solid Phase

Soil Constituents and Texture

The solid phase consists of (1) primary particles derived from thenonweathered rocks and deposits from which the soil is developed; (2) sec-ondary minerals (clays) that are electrically charged, derived from weath-ered primary particles (Marshall, Holmes, and Rose, 1996); and (3) organicmaterials that consist of fully and partly decomposed organic residues andplant parts such as roots, fungal mycelia, and decomposed fauna.

Soil Structure

Solid soil particles of various origins, properties, and sizes, mixed in var-ious proportions, define the textural soil types (e.g., sandy soils, clay soils).The solid soil particles are spatially arranged in various skeletal matricesthat exhibit certain structural hierarchies (Tisdall, 1996; Tisdall and Oades,1982; Hadas, 1987b; Dexter, 1988). These hierarchies are made of struc-tural subunits of various sizes, in a variety of spatial arrangements, and havecomplex pore networks within and between the particles. In these pores, airand soil solution are found in varying proportions. The structural hierarchyfollows a general pattern in which the smallest basic units, clay domains ortactoids (1 to ~20 m in size) made of clay particles, are joined together bycation bonds, electrical attraction, and/or organic cements. These domainscombine with larger particles and organic cementing substances to formmicroaggregates (50 to 200 m in size) which, in turn, form larger units and

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so on (semi- and macroaggregates, clods, and blocks). The larger the soilunit is the coarser are its pores and the greater the number of interunit fis-sures. These structures recall the internal arrangement of smaller, denserunits encapsulated in larger, more open ones (Tisdall and Oades, 1982;Oades and Watts, 1991). The pores are smallest in diameter within the do-mains and largest among the largest structural units. Soil structures, formedunder natural conditions by wetting and drying, freezing and thawing, andswelling and shrinking cycles, or formed artificially by tillage operations,show great variability (Dexter, 1988, 1991; Marshall, Holmes, and Rose,1996; Hadas, 1997).

Soil structure determines both total soil porosity and pore size and con-nectivity distributions. Intraparticle cohesion and interparticle adhesion arelargest within and between the clay domains, and both decrease as the sizeand complexity of the structural units increase, because of the diminishingnumber of interparticle contact points and the increasing number of fissuresand cracks. The structural stability of such a complicated matrix dependsgreatly on moisture content (which weakens cementing bonds and electri-cal attraction), internal stresses (caused by swelling, water surface tension,entrapped air pressure, and overburden), and external loads (vehicular traf-fic and animal tracking). Under these stresses soil structures will deform,fail, or collapse (slaking, compaction, seal forming) if the bonding forces(cohesion and adhesion) are weaker than the loads imposed on the structure(Hadas, 1987b; Dexter, 1988; Marshall, Holmes, and Rose, 1996). Bothpore volume fraction and pore size distributions are closely related to water,thermal, and aeration regimes, as well as to the soil mechanical propertiesof natural soil environments or artificially produced seedbeds. Soil struc-tural changes in response to climatic conditions (rain, freezing, and thawingevents) or human activities (irrigation, tillage operations, compaction)cause great variations in soil density, total soil porosity, and pore size distri-bution and thus affect the water, thermal, and aeration regimes and soilstrength (Marshall, Holmes, and Rose, 1996; Hadas, 1997).

Obviously, soil structure and its stability are of great importance to seedgermination. Seeds may fall into natural fissures and cracks or be sown inbetween crumbs formed by tillage. Some may germinate; others may be en-trapped by unstable, slaking structures or within fissures closed by swellingsoil and their germination may be delayed or inhibited, or they may reentersecondary dormancy (Egley, 1995).

Soil Mechanical Behavior, Soil Crusts, and Soil Compaction

Soil mechanical behavior is determined by the intra- and interparticlebonds (cohesion and adhesion, respectively) which become stronger as the

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spacings between soil particle and unit diminish (i.e., as soil density in-creases). These forces are manifested in soil resistance to shear by tillageimplements, compressibility under vehicular loads, impedance to penetra-tion by fine needles, and tensile resilience. Soil impedance affects seed wa-ter uptake, thus, in turn impairing seed germination and stand establish-ment. Seed mechanical resistance can also diminish stand establishment byaffecting elongation of radicles and roots and the emergence of coleoptilesand hypocotules through soil crusts (Bowen, 1981; Hadas and Stibbe, 1977;de Willingen and van Noordwijk, 1987; van Noordwijk and de Willingen,1991; Unger and Kaspar, 1994; Marshal et al., 1996). Soil structure disinte-gration and slaking caused by fast soil surface wetting (because of low soilstructural stability, raindrop impact, fast wetting, and implosion by en-trapped air) and the subsequent formation and densification of soil seals re-duce water infiltration and aeration. These crusts greatly impede seedlingemergence, and this impedance increases as they become denser and drier(Bolt and Koening, 1972; Hadas and Stibbe, 1977; Dexter 1988; Bradfordand Huang, 1992; Morin and Winkler, 1996).

Soil compaction results in soil densification caused either by shrinkageor external loads. Compaction, therefore, reduces the total soil porosity,pore size, gaseous exchange, and water infiltration, and increases soil im-pedance to penetration, impairing water spatial distribution and restrictingseed germination and seedling establishment (Bowen, 1981; Hadas, Larson,and Allmaras, 1988; Gupta, Sharma, and De Franchi, 1989; Unger andKaspar, 1994; Horn et al., 1994). Complete alleviation of an impaired soilphysical environment depends on the processes that led to that impairment.The deleterious effects of crusts are rather easily alleviated by delicatelyfragmenting the newly formed crust, but complete rehabilitation of proper-ties of compacted soil is almost impossible; great energy inputs are requiredto break up the dense soil into a favorable seedbed. Such efforts usually re-sult in coarser seedbeds, improper stands, and lower yields (Hadas, Wolf,and Rawitz, 1983, 1985; Hadas et al., 1990; Wolf and Hadas, 1984).

Water Regimes In Soils

Water Content

In an air-dry soil, a minute amount of water is adsorbed on soil particles(hygroscopic water content), whereas in a saturated soil the pore system iscompletely filled with water. The water-filled volume fraction of the soil,termed the volumetric soil water content, w, varies widely, especially in theupper soil layer. These variations depend on climatic and environmental

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conditions (e.g., rain, evaporation, drainage, vegetation, and human activity(irrigation). Integration of w with respect to depth gives the total wateramount held in the soil to a given depth. Periodic integration of w with re-spect to soil depth leads to estimates of the soil water balance, i.e., theamounts of water added to or withdrawn from a given soil volume. Quanti-tative predictions of water movement into, within, and out of the soil can bederived from knowledge of the soil water energy status, i.e., the soil waterpotential, soil, the water transport properties of the soil, and the appropri-ate physical equations governing water movement in soil.

Soil Water Potential

Various forces act on water adsorbed or held in the soil pores (e.g., gravi-tational, hydrostatic, matric forces derived from soil surface-water-air inter-actions, osmotic forces, and swelling forces derived from soil clay-water in-teractions). The influence of each of these forces, or their combinations, onsoil water is given by the amount of work that must be done when a minuteamount of water is transferred from a reference pool of water to the soil.That amount of work is termed the soil water potential (Kutilek and Niel-sen, 1994; Marshall, Holmes, and Rose, 1996).

The soil water potential, soil, is the algebraic sum of the several specificsoil water potentials derived from the various forces acting on soil water. Itis given in Equation 1.1, where g, p, e, m, and os are the gravita-tional, hydrostatic, envelope (overburden, mechanical constraint), matric(derived from the adsorbed, interfacial soil-air-water tension), and osmoticsoil water potential components, respectively. The gravitational and hydro-static components ( g and p) can be ignored when one deals with germi-nating seeds affected by a small volume of nonsubmerged, moist soil sur-rounding them (Equation 1.1a).

soil = m + e + os + g + p (1.1)soil = m + e + os (1.1a)

When a soil is either saturated or submerged in pure water, soil has neg-ative values relative to pure water under the same conditions. In practicalterms it means this water uptake by seeds or roots from unsaturated soil iscarried out at the expense of metabolic energy. The osmotic component,

os, varies with salt concentrations and compositions, clay content, andclay type and requires a semipermeable membrane separating the soil waterfrom the seed cells. The matric component, m, exists in unsaturated soilsand depends on water content, soil pore size, distributions, and soil struc-

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tural stability. The relationship between w and m is known as the soilcharacteristic or retention curve. Assuming the soil pores resemble bundlesof capillary tubes (Marshall, 1958, 1959), the first pores that will be drainedunder minute matric forces will be the large ones (the interaggregate pores,or fissures and cracks). As m decreases, the smaller pores drain, with thenarrowest pores (in the clay domains in which water is held by very strongmatric forces) draining last. Upon wetting, the filling order of pores withwater is the reverse of the draining order. Soil water characteristics curvesare not unique and depend on the way they were obtained, either by drain-ing a saturated soil or by wetting an unsaturated or an air-dry soil (Kutilekand Nielsen, 1994; Marshall, Holmes, and Rose, 1996). This phenomenonis called soil water characteristic hysteresis. The measured m for a given

w will be lower for the draining characteristic curve than for the wettingone. This discrepancy results from irregular pore cross sections, bottlenecksconnecting pores of differing radii, and smaller wetting angles than at drain-ing (Kutilek and Nielsen, 1994; Marshall, Holmes, and Rose, 1996). Inpractical terms, this means that a seed embedded in a moist soil will startimbibing at a given value of m, which will decrease instantaneously be-cause of the abrupt change from wetting to draining characteristics. Thesevariations in m will be further aggravated if the seeds are placed in aggre-gated beds in which wide hysteretic variations are to be expected and arepartly explained by the pore exclusion principle (Amemiya, 1965; Mar-shall, Holmes, and Rose, 1996).

Swelling Soils and Collapsed Soil Structure Matric Potential

During fast wetting, the soil structure deteriorates and breaks down(slakes), the structural fragments are reorganized, and, as in the case of claysoils, the soil undergoes volume change upon wetting or drying. In thesecases, the pore system and the water characteristics of the soil change (Mar-shall, Holmes, and Rose, 1996). Upon draining, swollen or slaked soils re-main saturated, although an appreciable volume change may be observedand m becomes negative. Thus, seeds embedded in swollen or slaked soilmay be subjected to oxygen deficiency during germination. Seeds germi-nating under external load or caught in a drying clay or compacted soil maybe adversely affected by the confining pressure the external load or shrink-ing material may exert on them, reducing their ability to take up water andgerminate (Collis-George and Williams, 1968; Hadas, 1985).

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Water Movement in Soils

Water is forced to move in soil when there is a driving force resultingfrom a water potential gradient between two points in the soil or betweenseed and soil water. The rate of movement depends on the prevailing waterpotential gradient and the water conductivity of the matrix in which watermovement occurs (e.g., seed or soil). As the water moves, the water contentof a given soil volume may be depleted, remain unchanged, or increase. Ageneral quantitative description of water flow, which accounts for watercontent variations (law of conservation of matter) that account for water po-tential and water content variations, is given in Equation 1.2, where qw is theinstantaneous water flux and K is the soil water conductivity. In saturatedsoil it will be termed the soil hydraulic conductivity, Ks, and in an unsatu-rated soil it is termed the soil capillary water conductivity, K( w). Ks de-pends on the soil pore-size distribution, pore connectivity, and the total wa-ter content, whereas K( w) depends on soil pore-size distribution and poreconnectivity within the water-filled soil volume fraction,

/ x(qw) = w/ t = / x[K( w) soil/ x] = / x[D( w) w/ x] (1.2)

where ( w/ t) is the time variation of the volumetric water content andD( w) = K( w)[ m( w)/ w] is the soil diffusivity to water, and [ ( w)/ w]is the specific water yield or specific water capacity (Kutilek and Nielsen,1994; Marshall, Holmes, and Rose, 1996). In aggregated beds Ks increaseswith increasing aggregate size, but as the soil matric potential decreasesK( w) increases as the aggregate size decreases (Amemiya, 1965).

As water is removed from an unsaturated soil, w may change, some-times causing changes in m and os and in the soil capillary conductivityto water, K( w). These changes depend on the water amounts taken up andin which mode (wetting or drying). Solutions for Equation 1.2, derived forparticular cases, e.g., water flow to seeds or roots, will be given and dis-cussed in the following.

Temperature Effects on Soil Water Characteristicsand Transport in Soils

Soil water properties, i.e., water characteristics, conductivity, and diffus-ivity, are temperature dependent. Water characteristics are affected mostlyby temperature because of changes in water surface tension and volumechanges of entrapped air bubbles. The soil water matric potential decreasesas the temperature increases, but changes observed in dry or saturated soils

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were smaller than those in moist to wet soils (Taylor and Stewart, 1960;Chahal, 1965). Water conductivity and diffusivity are affected by tempera-ture-related changes in water viscosity and vapor diffusion, condensation,and evaporation in pores (de Vries, 1958, 1963). Temperature gradientscause water, in both liquid and vapor phases, to move from high to lowertemperature zones in the soil (Philip and de Vries, 1957).

In moist soils the diurnal temperature wave will tend to reduce soil waterloss to the atmosphere during the day by forcing the water to follow the heatwave into the soil. During the night the direction of water movement is re-versed and losses to the atmosphere increase for a few days (Hadas, 1975)while the water is in the soil surface layer. Diurnal variations in water con-tents due to deposition and evaporation of water vapor condensation havebeen observed (Rose, 1968; Hadas, 1968; Jackson, 1973) and affect germi-nation, as suggested by Collis-George and Melville (1975) and Wuest,Albrecht, and Skirvin (1999).

Soil Thermal Regime

The radiant energy, intercepted at the soil surface, governs the thermalregime of the soil. Its measure depends on latitude, land slope and relief,soil color, and vegetative cover (van Wijk, 1963). A rather small amount ofthe intercepted radiative energy heats the soil; a fraction of it is reflected,another fraction is reradiated as infrared radiation, a fraction directly heatsthe air in contact with the soil surface, and the rest is dissipated as latent heatby evaporating soil water. The soil heat flux, G, depends strongly on thethermal properties of the soil, namely soil heat capacity and thermal con-ductivity, which vary with soil texture, structure, bulk density, and watercontent (Buckingham, 1907; van Duin, 1956; van Wijk, 1963; de Vries,1963).

Diurnal and annual radiation patterns result in diurnal and annual heatwaves. The amplitude and phase shift of the diurnal wave strongly influencegerminating seeds through (1) variations in Q10,* the rate of biological pro-cesses; (2) changes in the level of the competition for available oxygen withthe surrounding microbiota; (3) changes in soil water properties (osmotic,matric and potentials, water conductivity, and diffusivity); and (4) coupledthermal, liquid, and vapor transport processes, vapor condensation, andevaporation (Philip and de Vries, 1957; de Vries, 1963; Hadas, 1968;Kutilek and Nielsen, 1994).

*Q10: a factor for the change in reaction rate for a 10°C temperature increase

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Volumetric Heat Capacity

The volumetric heat capacity of a soil, C, depends on the volumetric con-tents of the soil constituents, and their specific heat capacities and can becalculated from the sum of the respective products (the volumetric contentof each soil constituent by its specific heat capacity). The greater the volu-metric water and/or the solid fractions, the greater the soil volumetric heatcapacity (de Vries, 1963). A larger volumetric heat capacity means that agreater amount of heat will be required for a given temperature increase ofa given soil volume. In practical terms this means that a dry seedbed with alow volumetric heat capacity will tend to reach high temperatures duringthe day and low temperatures in the later part of the night. Such fluctuationsexpose young seedlings to risks of sun scorching during the summer and offrost damage in the early spring. These variations can be greatly moderatedby increasing the volumetric heat capacity by increasing w through irriga-tion and/or by compacting the soil.

Thermal Conductivity

The soil thermal conductivity, , depends strongly on soil constituents,i.e., the solids, air, and water. Whereas air is a poor conductor, the solid par-ticles and water are good conductors; the heat conductivity of soil solids isfour to five times greater than that of water, which is in turn about three or-ders of magnitude greater than that of air. In saturated soils and air-dry soilsonly two constituents contribute to , i.e., solids and water and solids andair, respectively. The thermal conductivity of a moist, unsaturated soil de-pends on all three constituents and can be calculated from their volumefractions, particles shape, and their respective thermal conductivities, assuggested by de Vries (1963), or it can be measured (van Wijk, 1963). Thesoil thermal conductivity increases as the solids and water volume fractionsincrease, due to better interparticle contacts.

Heat Transfer

The generalized heat transport relationship in soils is derived from Fou-rier’s law, G = – dT/dx, in which G is the soil heat flux, dT/dx is the temper-ature gradient, and is the effective thermal conductivity. Under naturalconditions in which the temperature of the soil surface varies constantly, thegeneral heat transfer that takes account of temperature changes with time(law of energy conservation) is given, for a certain depth z, in Equation 1.3,

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in which ( T/ t) is the rate of temperature change with time at a given pointin the soil,

( T/ t) = / z[ / c( T/ z)] = / z[ T/ z] (1.3)

where ( 2T/ z2) is the rate of change of temperature gradient with respect todistance, is the effective thermal conductivity, c is the volumetric heatcapacity, and = / c is the thermal diffusivity of the soil (Marshall,Holmes, and Rose, 1996).

Diurnal and Annual Temperature Cycles in Soils

Although the diurnal and annual heat waves appear as a single, com-pound wave, it is possible to distinguish between the cycles by assuming thesoil surface temperature to follow two different sinusoidal waves. The soiltemperature dependence on time, t, and depth, z, for a homogeneous soil isgiven in Equation 1.4, where T(z, t) is the soil temperature, = 2 / , is theperiod (day, year), and A0 and Aav are the amplitude and mean temperatureat the surface, respectively (Carslaw and Jaeger, 1959; Kirkham and Powers,1972).

T(z,t) = Aav + A0exp – {( /2 )1/2z}sin{ t – ( /2 )1/2z} (1.4)

The exponential term signifies the decay with depth of the temperatureamplitude, and the argument of the sine term yields the lag between thetimes at which maximal or minimal temperature is reached at the soil sur-face and at depth z. The diurnal wave penetrates to a depth of 15 to 35 cm,and the annual wave to as much as 6 m, depending on the soil thermal prop-erties. Normally, sown seeds are confined to the shallow layer below the soilsurface, where they will be subjected to temperature amplitudes almostequal to those at the soil surface with a minimal time lag. Seeds sown intoan aggregated seedbed may be scorched by extreme midday temperatureamplitudes during the summer or be exposed to freezing hazards during lateautumn and early spring. Kebreab and Murdoch (1999a) reported that in-hibitory effects of temperature on germination were more evident underfluctuating than under constant temperatures; they found that the effect ofhigh temperatures on germination was greatly influenced by the amplitudeand thermoperiod of fluctuating temperature (Kebreab and Murdoch, 1999b;Stout, Brooke, and Hall, 1999).

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Temperature Cycles in Layered Soils and Seedbeds

The temperatures around seeds are greatly affected by the aggregate sizedistribution, water content, and the existence of soil seals. Under natural sit-uations, the soil thermal properties vary with time and soil depth; therefore,the heat wave becomes more complex with depth, and this is especially truein seedbeds in which great variations in soil structure and layering existwithin short horizontal and vertical distances. These variations result inlarge temporal and spatial variations of , c, and ; therefore, the thermalregimes of these soils cannot be predicted by simplified analytical equa-tions as given previously (Hadas and Fuchs, 1973). Peerlkamp (1944), vanDuin (1956), and van Wijk and Dirksen (1963) developed analytical modelsfor predicting the changes in soil temperature in layered soils. However,where the soil properties and structure vary continuously, the analytical so-lutions fail and predictions of changes in soil temperature in layered soilsrequire the use of computers and complex computer programs. Neverthe-less, some conclusions can be drawn from the previous analysis. When anaggregated bed or a layer of dry soil lies over dense, moist soil, the soil sur-face temperature fluctuations will show increased amplitudes and the heatwave penetration will be shallower. This theoretical finding supports thepractice of using dry mulches on the soil, either to keep the soil cooler in thesummer or insulate it from cold in the late autumn.

Temperature Dependence of Q10 Coefficient

The diversity in the effects of temperature on germinating seeds causedby variations in the Q10 coefficient of enzymatic reactions is rather compli-cated. Q10 varies between 1.5 and about 3 for productive and synthesis reac-tions (which means that for each 10°C rise in temperature, the reaction rateincreases by a factor of 1.5 to 3) and may be as high as 6 for denaturizationprocesses (Voorhees, Allmaras, and Johnson, 1981). The Q10 biological re-action rate coefficient, defined by R1/R2–Q10

[(T2–T1/10)], is derived from anonlinear relationship and its accumulated effect under fluctuating temper-atures may be greater by 15 to 40 percent than that calculated from the Q10value for the constant, mean temperature. This explains why seeds under afluctuating temperature regime germinate faster then those under the con-stant mean temperature.

Aeration Regime

Water-free voids in the soil matrix contain mixture of gases, the propor-tions of which change with depth, temperature, root and microbial activity,

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water content, and void connectivity. Gaseous exchange occurs through air-filled pores and across water films. When the soil is air-dry the pore volumeis air filled and free exchange is possible, whereas in a saturated soil, al-though the pores are water filled, some air bubbles are entrapped. Near thesoil surface the soil air composition is very similar to that of the outer atmo-sphere (~79 percent N2, 21 percent O2, 0.03 percent CO2, and other gases)(Marshall, Holmes, and Rose, 1996). Normally, the oxygen concentrationdecreases with soil depth while the concentrations of CO2 and ethylene in-crease. During gaseous exchange, oxygen moves into the soil while CO2and ethylene move out of the soil. This exchange combines mass exchangedriven by soil temperature and/or barometric pressure-related soil-air ex-pansion, wind gusts at the surface, air displacement by rain or irrigation wa-ter, air entry resulting from soil desiccation, and gaseous diffusion. Diffu-sion is the most important gaseous exchange process in soils (Buckingham,1904; Rommel, 1922; Marshall, Holmes, and Rose, 1996). Wind-generatedair turbulence, which enhances diffusive exchange, is most effective in ag-gregated soil, where the gas exchange flux can be as high as 100 times themolecular diffusion flux, provided there are no seals (Farrell, Greacen, andGurr, 1966; Farrell and Larson, 1973). For germinating seeds found next tothe surface, oxygen supply by molecular movement, i.e., diffusion throughthe soil surface and within the air-filled soil pores, is of great importance.Seeds take up oxygen only after it has crossed the water films surroundingthem under optimal situations, or after it has passed through water films,water-filled pores, or even saturated seals when adverse conditions prevail.

Molecular diffusion is described by the first Fick equation, in which theinstantaneous oxygen flux, qox is driven by the oxygen gradient, dC/dx, andDef is the effective soil-air diffusion coefficient (Equation 1.5).

qox = –Def dC/dx (1.5)

In a medium such as soil, in which diffusion may occur only through in-terconnected air-filled pores or water films, Def equals a complex meanweighted value combining its value in water Dw and the effective continu-ous air-filled pore volume fraction (Buckingham, 1904; Currie, 1961, 1983,1984; Currie and Rose, 1965; Marshall, Holmes, and Rose, 1996). Varioussimple relationships have been derived for the Def/Dair, e.g., Def/Dair = b air(Pennman, 1940), or as power functions of air (Marshall, Holmes, andRose, 1996). These relationships point out the wide variations in Def with

air. (volumetric air content). Greenwood (1975) and Wesseling (1974)have shown that for a volumetric water content w 0.10 or 0.12, respec-tively, pore connectivity disappears and diffusion will occur only through

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the water in the soil pores. The oxygen diffusion coefficient in water issmaller than that in air by a factor of 104; therefore, it may well be that theoxygen supply rate to germinating seeds imbedded between moist soilcrumbs in a well-prepared seedbed is controlled only by the water film cov-ering the seeds. However, the O2 supply in compacted or sealed soil will bereduced, because of the low porosity and high water content, respectively.The degree of impairment will be greater if, in addition to the factors justmentioned, the temperature is high, so that high biological activity will beenhanced (Glinski and Stepniewski, 1985; Glinski and Lipiec, 1990).

SEEDBED PREPARATION, CHARACTERIZATIONOF SEEDBED ATTRIBUTES, AND SEEDBED ENVIRONMENT

CONDITIONS AND SEED GERMINATION

The seedbed is the finely tilled, loose topsoil layer especially prepared toensure fast, uniform germination and emergence into which seeds are sown(Keen, 1931; Slipher, 1932). Seedbed preparation requires a sequence oftillage operations aimed at fragmenting the bulk soil, manipulating the dis-turbed soil structure and improving soil tilth; it provides favorable air,water, and heat regimes and reduces mechanical resistance to seed germina-tion, emergence, and root development (Slipher, 1932). Great importance isgiven to specifying and then attempting to produce a desired seedbed(Braunack and Dexter, 1989a,b; Dexter, 1991; Hadas, 1997); nevertheless,all the great effort, labor, and equipment invested in producing a specifiedseedbed may be wasted. The seedbed may, under optimal conditions, com-plete its usefulness within few days after sowing, once seedlings are estab-lished, but under the impact of adverse weather the soil structure may fail orcollapse because of fast wetting and drop impact. As a result, the failedstructure may impose mechanical constraints on seed germination andstand establishment.

Seedbed Preparation and Seedbed-Characterizing Indices

A bulk soil structure that has settled during previous seasons has to befragmented and modified by one or more tillage operations and rearrangedinto a seedbed made of layers of aggregates that vary in their size ranges(Ojeniyi and Dexter, 1979a,b; Hadas and Shmulewich, 1990). Purposelymodifying a soil bulk structure to form a desired seedbed is always a matterfor compromises, aiming to minimize the risks of failures of seed germina-tion, emergence, and stand establishment under anticipated future weatherconditions, soil structure variations, and resultant seedbed environmental

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conditions, while reducing energy and labor investments. For each combi-nation of soil, soil structure, structural stability, local climate uncertainties,and crop, several possible compromise solutions exist, derived by trial-and-error procedures (Hadas, 1997; Hadas, Wolf, and Meirson, 1978; Hadas,Wolf, and Stibbe, 1981; Unger, 1982).

Under arid conditions, the establishment of a uniform stand requiresgood seed-soil-water contact to ensure rapid water uptake and seedlingemergence and to avoid the effects of fast soil-surface drying and crusthardening (Hillel, 1960; Hadas, 1997; Hadas and Stibbe, 1977). These re-quirements dictate a seedbed made of small, fine aggregates with narrowsize distribution, so as to ensure good seed-soil contact and low evaporativelosses (Russel, 1973; Hadas, 1975; Hadas and Russo, 1974a,b). However, afinely aggregated seedbed presents increased crusting hazards upon wettingand drying (Hadas, 1997). Under wet conditions, improved drainage, aera-tion, and enhanced soil warming in cold regions are sought, which stimulateefforts to produce coarse tilth and to shape the soil surface as ridges orbenches to improve drainability and increase air-filled porosity. However,these latter benefits will be balanced by reduced heat capacity and thermalconductivity, which increase the risks of enhanced temperature variationsnear and at the soil surface in hot regimes, or even those of freezing in coldregimes (van Duin, 1956; van Wijk and Dirksen, 1963).

Soil Tillage and Seedbed Formation

Tillage implements exert external stresses on the soil bulk causing it tofail in several different modes (brittle, shear, compressive, and plastic defor-mation), depending on initial soil conditions (bulk density, water contentand existing fissures, cracks, root channels), tillage implements, type, andmodes of operation. The extent, mode, and fineness of soil failure or frag-mentation determine the need for further tillage work and ultimately thequality of the produced seedbed.

Field soils that are compacted and tilled periodically consist of neatly ar-ranged soil clods, macroaggregates, and blocks, differing in their size den-sity and crack networks (Hadas, 1997). When tilled, soil units are separated,torn, or fragmented and moved sideward and upward. In dry soils, severaltillage implements are applied sequentially to fragment the soil and pro-duced the desired seedbed tilth (Hadas, Wolf, and Meirson, 1978). Thenumber of tillage passes required diminishes as soil water content nears thatof the plastic limit water content, which is approximately that of wiltingpoint in many soils (Dexter, 1988, 1991; Hadas and Wolf, 1983; Hadas,Wolf, and Meirson, 1978). Current knowledge of soil fragmentation pro-

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cesses is very limited; therefore, exhaustive tillage trials are required to ob-tain the resulting soil fragmentation data (Dexter, 1977; Koolen, 1977;Hadas, Wolf, and Meirson, 1978; Hadas and Wolf, 1983; Gupta and Larson,1982; Perdok and Kouwehoven, 1994; Guerif et al., 2001; Young, Craw-ford, and Rappoldt, 2001). The total inputs of energy and labor in tillingsoils depend on (1) soil conditions and constituents (density, water content,fissures, pores and failure plane nets, surface energy of soils), (2) types ofimplements used, and (3) the soil structure fineness required (Gupta andLarson, 1982; Hadas, 1987a, 1997; Hadas and Wolf, 1983; Hadas, Wolf,and Meirson, 1978; Wolf and Hadas, 1987; McPhee et al., 1995; Royten-berg and Cheplin, 1995; Perfect, Zhai, and Belvins, 1997). It becomes obvi-ous that any proposed seedbed preparatory procedure must be based on ahuge database and must be formulated along a delicately balanced, com-pound probabilistic approach. That approach has to account for (1) annualand seasonal weather variability; (2) known probabilities of attaining theright tilth by using the right implements in the proper order and the best pos-sible soil conditions; (3) soil tilth stability and probability of seedbed failurecaused by weather events, traffic, etc.; and (4) known characteristics andbehavior patterns of seed lots. In the light of the complexity of the processesinvolved, our current knowledge gaps, and our inability to assess the proba-bilities of the various system components, such an approach eludes us andany simple seedbed modeling and forecasting of its properties is precluded(Hadas, Wolf, and Meirson, 1978; Hadas, Larson, and Allmaras, 1988;Kuipers, 1984; Lal, 1991; Hadas, 1997; Guerif et al., 2001).

In order to standardize seedbed preparation, some physical indices, char-acterizing the preferred soil seedbed structure to be obtained, have to be de-fined, tested, and accepted as recognized and official indices. Suggestedphysical determinations of seedbed characteristics have appeared and havebeen discussed in the literature. The characteristics addressed included totalporosity, aggregate size distribution, shear strength, infiltration rate, sorp-tivity, aggregate stability to water and wind abrasion, and resistance to pen-etration (Russell, 1973; Hadas and Russo, 1974b; Tennent and Humblin,1987; Braunack and Dexter, 1989a,b; Thurburn, Hansen, and Glenville,1987; Christiansen, Foley, and Glanville, 1987; Collis-George and Lloyd,1979). The procedures to determine these indices have also been described(Hadas, Wolf, and Meirson, 1978; Tennent and Humblin, 1987; Braunackand McPhee, 1991), but so far none of the indices have been recognized asofficial indices, probably because their determinations are cumbersome anddemand much time and labor, and once determined they may change in aninstant by rainfall, irrigation, or traffic.

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Seedbed Aggregate Size Distribution and Seed Germination

The most commonly used seedbed-defining index is the size distributionof the aggregates found at the seed placement depth. Russell (1973) hassuggested that a seedbed consisting of aggregates larger then 0.5 mm butsmaller then 5.0 to 6.0 mm will provide the ideal conditions for seed germi-nation and emergence. In general, smaller aggregates reduce soil waterevaporation and soil drying (Holmes, Greacen, and Gurr, 1960; Farrell,Greacen, and Gurr, 1966; Kimball and Lemon, 1971; Hadas, 1975). Nasrand Selles (1995) used two logistical models to predict wheat seed germina-tion and concluded that final emergence rates were negatively affected byseedbed density and aggregate size. Beds made of 0.5 to 3.0 mm aggre-gates, 3.0 to 10.0 cm deep, were reported to maintain minimal water losses(Hillel and Hadas, 1972; Hadas, 1975). These seedbeds have to be preparedprior to soil wetting (Hillel and Hadas, 1972; Allmaras et al., 1977). Hadasand Russo (1974b) stated that a seedbed should consist of aggregatessmaller than one-fifth to one-tenth of the seed size if seed-soil contact is thegoverning factor in water uptake and is crucial to seed germination. Theserecommendations seem to be right for medium to large seeds but fail whensmall seeds placed next to the soil surface are considered (e.g., celery, car-rots, sugar beet). Under these situations, soil water content, aggregate sizedistribution, and good seed-soil contact are the germination controlling fac-tors (Hadas and Russo, 1974b); frequent irrigation keeps the soil moist, im-proves seed-soil contact, and reduces the mechanical impedance of sealsthat may be formed.

Soil Surface Relief—Ridged Seedbed

Shaping the seedbed into ridges is a common practice for overcomingpoor stand establishment on poorly drained soils or under low spring tem-peratures. Sowing on ridges enables the seedbed temperature on ridges torise by 2 to 3°C above that of a flat seedbed, which promotes emergence inwet, cool regions (Spoor and Giles, 1973; Gupta et al., 1990). Ridging im-proves the utilization of winter-stored soil water by summer field crops andallows sowing a few days earlier than on flat seedbeds and thus enablescrops to avoid pests (Hadas and Stibbe, 1973; Tisdall and Hodgson, 1990).However, yields on a flat seedbed may be the same as or higher than yieldson ridges.

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WATER UPTAKE BY SEEDS AND SEEDLINGS

Water uptake by seeds is an essential step toward rehydration of seed tis-sues and initiation of the metabolic processes in seeds, and the minuteamounts of water required for germination depend on the seed genome andits individual constituents. The various organs (e.g., embryo, cotyledons)and tissues differ in their internal physical structure, biochemical proper-ties, and chemical composition; therefore, they may differ in their water re-tention, distribution, and swelling properties (Stiles, 1948; Bewley andBlack, 1982; Koller and Hadas, 1982).

Water uptake by dry seeds is characterized by three phases, controlled byone of of the following factors: (1) the seed properties with respect to water(e.g., seed water potential, diffusivity to water), (2) the soil-water properties(e.g., soil-water potential, diffusivity, and conductivity to water of the soilaround the seed), and (3) the hydraulic properties of the seed-soil interface.

The initial phase in water uptake, the imbibition phase, is characterizedby a saturation kinetics pattern, depending on soil-seed contact, seed com-position, and the seed coat geometry and properties (Hadas, 1982). The sec-ond phase, the transition phase, is characterized by a low to negligible wateruptake rate. The third phase, the growth phase, is characterized by a rapid,exponential increase in the water uptake rate, accompanied by the emer-gence of the radicle. The first two phases are observed in dead, inert, and vi-able seeds alike, whereas the growth phase is unique to viable, germinatingseeds.

The Imbibition Phase

The imbibition phase, usually considered to be a passive one, starts withentry into the seed of water, which is distributed in crevices, cracks, andflaws in the seed cover and tissues and is absorbed by the seed colloids. Wa-ter uptake rate measurements toward the end of this phase have shown theserates to be temperature dependent and accompanied by observed increasesin respiration rate and light sensitivity in some seed species (Pollock andToole, 1966; Taylorson and Hendricks, 1972; Tobin and Briggs, 1969;Karssen, 1970; Berrie, Paterson, and West, 1974). These observations sug-gest that water uptake during imbibition is not passive at all but instead be-comes an active process at a rather early stage of this phase. The end of theimbibition phase is generally marked by an asymptotic approach to a finalwater gain. The rate of approach to the final value of water gain and its valuedepend on soil water potential, soil hydraulic properties, and seed composi-tion (Hadas, 1982; Bradford, 1995).

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The Transition Phase

During the transition phase, also known as the pause phase (Haber andLuippold, 1960), the seed moisture content, respiration rate, and apparentmorphology remain unchanged. Nevertheless, a variety of metabolic pro-cesses are activated (Koller and Hadas, 1982), and differences in activitylevels of processes and the order of their occurrence have been observedamong seeds of various species and among seeds differing in their hydra-tion levels (Hegarty, 1978). Therefore, any adverse environmental condi-tions may lead to redrying of the seeds, so water stressing them and affect-ing their hydration levels may impair, retard, or even inhibit germination. Ifno damage resulted, no dormancy was induced, and no inhibitory processeswere triggered, germination of these seeds upon rewetting would be en-hanced due to the high concentrations of unused metabolite accumulatedprior to drying (Boorman, 1968; Koller, 1970). These are the basis for seedpriming, a technique known also as “chitting” (Hegarty, 1978) (see Chap-ter 4).

According to Bradford (1995), the transition phase can be considered asgermination, as its duration influences the initiation time and the extent ofradicle growth. Dormant seeds have been observed to reach the transitionphase and to remain in it for long durations that extend to weeks or more be-fore germination (Powell, Dulson, and Bewley, 1984; Bradford, 1995).

The Growth Phase

The growth phase starts with an increased respiratory rate, the initiationof cell division, and extension of the embryonic radicle cells and ends withradicle protrusion. The renewed water uptake rate depends on the water po-tential of the soil, adaptation of the seed water potential to soil environmen-tal conditions, and the seed-soil contact properties (Hadas and Stibbe, 1973;Hadas, 1982; de Miguel and Sanchez, 1992; Ni and Bradford, 1992; Brad-ford, 1995). As pointed out earlier, the distinction between the phases is anarbitrary partitioning of the continuous, sequential order of processes thatleads to germination. Actually, all the processes are interdependent and theinterrelationships between them suggest that each phase greatly depends onthe preceding phases, water uptake rates, and total water uptake (Hadas,1977a; Hegarty, 1978).

In order to generalize the observations and conclusions brought up previ-ously and to model germination in various seed-substrate systems, it is nec-essary to quantitatively define the physical properties of the substrate (e.g.,soil) and the seed and their interactions. Practically, fulfillment of such a re-

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quirement is almost impossible; instead, one reverts to simple indices orcharacteristics such as critical seed hydration level or critical water poten-tial (Hunter and Erickson, 1952).

It is generally accepted that to germinate, a seed must reach a minimalwater content known as the critical hydration level, defined as the minimalamount of water taken up by a seed that will induce germination (Hadas,1970; Koller and Hadas, 1982). It does not reflect the water distributionamong the seed components, nor does it have an absolute value since it de-pends on the water uptake rate, variations in external soil water content,temperature, and seed adaptation to variations in these factors (Koller andHadas 1982; Hadas 1982). The amount of water gained by the seed is theweighed mean water gains by the various parts of that seed. Blacklow(1973) has reported that whole corn seeds gained 75 percent of their initialweight, whereas the embryos, which form only 11 percent of the seed’sweight, gained 261 percent, and the endosperm, which forms most of theseed mass, gained only 50 percent. The critical hydration level concept, de-veloped for completely immersed seeds, fails in cases of partial seed wet-ting which occur when the wetted seed volume includes only the embryoand the adjacent storage tissues (Hydecker, 1968, personal communica-tion).

Critical water potential is defined as the external water potential value ator below which seeds cannot reach their critical hydration level. Fully im-bibed seeds can germinate and start growing even when the substrate or soilwater potential is still decreasing and is far below that critical value(McDonough, 1975; Bradford, 1995). Hunter and Erickson (1952) deter-mined critical water potential values of –1.25, –0.79, –0.66, and –0.35 MPafor corn, rice, pea, and clover seeds, respectively. Values of –1.52, –0.7,–1.2, –0.6, and –0.35 MPa were reported for sorghum, cotton, chickpea,pea, and clover seeds, respectively, by Hadas (1970), Hadas and Stibbe(1973), and Hadas and Russo (1974a,b). These values were determined un-der static equilibrium water potential conditions. In practical situations, inwhich external water potential varies with water uptake, soil evaporation, ordrainage, these values may change as well. Computed critical water poten-tial values of –1.4, –2.0, –0.45, –1.1, and –1.5 MPa, for corn, sorghum, clo-ver, cotton, and chickpeas were reported for dynamic situations and perfectseed-soil-water contact by Hadas (1970), Hadas and Stibbe (1973), andHadas and Russo (1974b). The values obtained for corn indicate that cotton,chickpea, sorghum, and corn seeds can probably germinate at lower criticalwater potentials than those observed for equilibrium conditions by Hunterand Erickson (1952) and Hadas (1970).

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SEED-SOIL WATER RELATIONSHIPS

Water transport of water into, within, and out of the soil domain and intothe imbibing seed depends on the soil water potential gradients and watertransport properties (conductivity and diffusivity to water) of the variousseed-soil system components (Marshall, Holmes, and Rose, 1996; Kollerand Hadas, 1982; Hadas, 1982).

The water potential of dry seeds is extremely low compared to that ofmoist soils (Hegarty, 1978; Hadas, 1982). Seeds brought into contact with amoist soil will start taking in water at once, at a rate that depends on the wa-ter potential gradient between the seed and the soil. The seed water poten-tial will increase in accordance with seed water characteristics, external wa-ter potential, seed storage materials, and ambient temperature (Mayer andPoljakoff-Mayber, 1989). Water will move first from the soil and then to theseed, and as the water uptake proceeds, water will be depleted from the soilfarther away from the seed. The rate and degree of depletion will depend onthe water flux into the seed and hydraullic properties of the soil-seed inter-face (Collis-George and Hector, 1966; Phillips, 1968; Hadas, 1969, 1970,1982; Hadas and Russo, 1974a,b; Hadas and Stibbe, 1973; Shaykewich andWilliams, 1971; Williams and Shaykewich, 1971). Changes in soil watercontent will induce changes in water potential gradients and water conduc-tivity, and the seed water potential and diffusivity to water will change aswell (because of seed metabolism and reconditioning of the seed mem-branes and seed coat).

Seed Water Potential

Air-dry seeds have an extremely low water potential, seed, ranging be-tween ~–50 and –100 MPa (Hegarty, 1978), but as the seed imbibes water,the water content of the seed organs and its water potential increase. Sincethe seed organs differ in their constituents and structure, their specific waterpotential characteristics will differ; nevertheless, the measured seed waterpotential, seed reflects equilibrium water potential of the whole seed. Thetotal water potential of a cell, cell, in each of the seed organs equals thealgebraic sum of the various water potential components, as given in Equa-tion 1.6, where cell, os, cell, m, cell, and T cell are the total, osmotic,matric, and turgor water potentials of the cells.

cell = os, cell = m, cell + T cell (1.6)

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The osmotic cell water potential, os, cell, reflects the osmotic potentialcontributions of the various cell constituents. It changes as the germinationprocesses progress and adapt to the changing soil near the seed. The seedmatric water potential, m, cell, reflects the matrical forces imposed on thecell water content by the cell wall structure and neighboring cells. Theturgor component, T cell, represents the counterpressure exerted by thestressed elastic cell wall structure in response to the os, cell and the swellingpressures of hydrated proteins and cell organelles. In general, cell has anegative value except in fully turgid cells. By changing the concentrationsof its constituents and by modification of its membrane activity and selec-tivity, a cell can regulate its water potential and its water uptake or loss.Therefore, the seed changes during the various germination phases and asan adaptive response to varying environmental conditions, e.g., soil salinityor soil drying (Hadas and Stibbe, 1973), T cell may well also change whenthe seed membranes leak to the environment (Simon, 1974; Hegarty, 1978).

Specific Effects of m, os, and e on Seed Germination

The two important soil water potential components, m and os, are di-rectly involved in water transport to germinating seeds. Seeds have been re-ported to respond equally to equal changes in these two components, pro-vided their membranes were intact and fully active (Ayers, 1952; Richardsand Wadleigh, 1952; Hadas and Russo, 1974a; Manohar and Heydecker,1974; Collis-George and Sands, 1962). Biological systems differ in theirtissue permeability to water and salts and in their susceptibility to salt toxic-ity (Uhvits, 1946; Collis-George and Sands, 1959; Wiggans and Gardner,1962; Bewley and Black, 1982; Meyer and Poljakoff-Mayber, 1989). Smallreductions in soil matric potential were observed to affect germination to agreater extent than equal or even greater reductions in the soil osmotic po-tential (Uhvits, 1946; Ayers and Hayward, 1948; Collis-George and Sands,1959; Wiggans and Gardner, 1962; Collis-George and Hector, 1966; Wil-liams and Shaykewich, 1971; Hadas and Stibbe, 1973; Hadas and Russo,1974a,b). This difference in response is due to the fact that a slight changein m involves a change in soil water content, with corresponding reduc-tions in both soil conductivity to water and seed soil contact (Sedgley, 1963;Collis-George and Hector, 1966; Hadas, 1970; Hadas and Russo, 1974a,b).An obvious corollary to that is that the critical water potential cannot al-ways be taken as the sum of these two components when germination in soilis considered. The reason for this is that the presence of selective mem-branes will exclude salts from the water taken in by the seed and leave themoutside the seed, so that os will increase and will not be directly measured.

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Values cited for critical water potential were determined under constant po-tential laboratory conditions; therefore, they might differ from those pre-vailing in real situations.

In natural situations, wetting of the soil by precipitation or irrigation willincrease w and m , decrease os, and improve environmental conditionsfor germinating seeds as long as aeration is not impaired. However, reduc-tion in w because of evaporation will decrease both m and os and mayenforce changes in seed and reduce the rate and final extent of germination(Hadas, 1976, 1977a,b). These responses will be further aggravated if seed-soil contact is impaired as well.

The possibility that the matric soil water potential affects germination byits direct contribution to the soil effective mechanical stress was examinedby Collis-George and Hector (1966) and by Collis-George and Williams(1968). Their data suggested that the mechanical effective soil stress re-stricts seed swelling or even inhibits embryo development. Others (Hadas,1970, 1977b; Shaykewich, 1973) found that under natural situations, nor-mal stresses induced in seedbeds are too small to confine seeds or to impairtheir germination. A dry seed initially develops swelling pressures of up to~400 MPa, but upon completion of imbibition the pressure decreases to~0.1 MPa (Shaykewich, 1973). These values far exceed the normal soilstresses found in the field to be around 0.12 to 0.34 MPa (Williams andShaykewich, 1971; Hadas, 1985). Observed poor germination in com-pacted soils and next to traffic lanes, or of seeds entrapped in shrinking soil,can result from greater mechanical constraints than those described earlierand imposed on the seeds.

MODELING SEED GERMINATIONAND SEEDBED PHYSICAL ATTRIBUTES

Any attempt to model seed germination should address the relationshipsbetween the time course of seed germination, germination rate, final germi-nation percentage, time lag in germination initiation, and the external fac-tors affecting germination singly or in combination. Moreover, the modelparameters should be quantifiable, have relevant biological significance,and be based on measured seed germination time patterns. Models shouldprovide some forecasting capabilities. Determination of the relevant modelparameters requires proper experimental procedures carried out under con-ditions that closely resemble actual conditions and that are aimed at mini-mizing uncertain results. Chapter 3 is fully devoted to analyzing existinggermination models that account for changes in both germination rate andgermination percentage in relation to environmental variables, mainly tem-

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perature and water availability. However, for completeness, modeling seedgermination under field conditions should include quantification of thechanging seedbed properties, the complex spatial and temporal variationsin the soil structure, and the seed reactions to these variations. The final partof this chapter discusses quantifying the variables involved in these pro-cesses and developing models on the basis of such quantifications to com-plement germination models such as those described in Chapter 3.

Modeling Water Flow in Seed-Soil System and Germination

The dynamics of water uptake by seeds can be quantitatively calculatedby applying solutions of Equation 1.2, the pertinent water potential gradi-ents and water conduction properties, and the appropriate boundary and ini-tial conditions to the system under considerations (e.g., determination ofDseed). In most reported experimental determination of seed water uptakedata, the experimental procedure used precluded dynamic forecasting basedon water flow. Furthermore, they do not permit any distinction to be madebetween specific effects on seed germination and those of changes in waterpotential components, conductivity or diffusivity to water, and seed-soilcontact area. Water transport within the seed and from the soil to the seedcan be simplified by (1) assuming seeds to resemble spheres or cylindersand (2) using water contents and mean weighted diffusivities to water as theflow equation parameters for solving Equation 1.2, (Phillips, 1968; Hadas,1970; Hadas and Stibbe, 1973; Hadas and Russo, 1974a,b). This choice be-tween using variations in water content and using mean weighted diffus-ivities to water allows one variable ( seed or soil) to be dropped and simpli-fies computational complexities (Crank, 1956). Water flow from the bulksoil toward a germinating seed involves (1) water flow in the soil toward theseed surface, (2) flow across the soil-seed interface, (3) flow across the seedcoat, and (4) flow into the seed itself (Phillips, 1968; Hadas, 1970, 1982;Koller and Hadas, 1982; Hadas and Russo, 1974a,b). To estimate waterflow in each subsystem of the seed-soil system, Equation 1.2 has to besolved for each of the system components by using the specific boundaryand initial conditions and the particular properties with respect to water(e.g., those of the seed, seed coat-soil, seed-soil interface)

Seed and Soil Diffusivities to Water

Several procedures, based on solving Equation 1.2 for the proper bound-ary and initial conditions, were used to calculate the seed mean diffusivityto water (for details see Phillips, 1968; Hadas, 1970; 1982). Reported seed

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diffusivity data for various seeds range from 1.5 × 10–5 to 1.6 × 103 m2/day(Phillips, 1968; Hadas, 1970; Shaykewich and Williams, 1971; Ward andShaykewich, 1972; Hadas and Russo, 1974b). The reported values of soildiffusivity to water range between 4 × 104 and 5 × 107 m2/day for air-dry tonear-saturated soils (Bruce and Klute, 1956; Rijtema, 1959; Kunze andKirkham, 1962; Doering, 1965; Amemiya, 1965). The values for soils arehigher than those reported for seeds. Hadas (1970) has shown that the seedradius increases as the amount of imbibed water increases. If seed swellingduring imbibition tests for determining seed diffusivity to water was ne-glected, Dseed was found to increase with increasing seed mean water con-tent (Phillips, 1968; Hadas, 1970; 1976, 1977a; Hadas and Russo,1974b;Ward and Shaykewich, 1972; Shaykewich and Williams, 1971). Collis-George and Melville (1975) used a solution of Equation 1.2 that accountedfor seed swelling and found that the mean diffusivity of wheat seeds to wa-ter was 74 m2/day, a value which was practically the same as that reportedby Ward and Shaykewich (1972), who used the simplified solution to Equa-tion 1.2. Using reported data of soil diffusivity to water, Hadas (1970)showed that for a seed which maintains a low water potential and an activemetabolic system, the soil can provide water to the seed at a greater ratethan that observed experimentally. These calculations strongly suggest thatseed water uptake and germination are controlled by seed coat imper-meability apart from the low seed diffusivity to water.

Seed Coat and Seed-Soil Interface Diffusivity to Water

Water flow from the soil into a seed crosses the soil-seed interfacial zone,which consists of the seed coat and the seed-soil contact zone. Seed-soilcontact is seldom perfect; therefore, a restriction is imposed on water flowfrom the soil into the seed. Seed coats vary in their permeability to waterand may be impermeable, partially permeable, fully permeable, or evenconditionally permeable in cases in which coat permeability varies in spotsaround the seed.

Seed Coat Permeability to Water

In general, seed coats are nonuniform in shape and roughness and pres-ent especially differentiated zones such as micropyle, hilum, chalaza, andareas covered with either hydrophilic or hydrophobic materials (Werker,Marbach, and Mayer, 1979). These variations in seed coat features (e.g.,structure, ports of water entry) and properties affect seed coat permeabilityto water, seed water uptake rate, and seed-soil contact impedance to water

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flow (Christiansen and Moore, 1959; Manohar and Hydecker, 1974; Stoneand Juhren, 1951; Quinlivan 1971). Morris, Campbell, and Wiebe (1968)reported seed coat permeability values ranging from 1.8 × 10–5 to 4.4 × 10–5 m/day for detached snapbean seed coats. Hadas (1976) calculated seed coatdiffusivity to water ranging from 3 × 101 to 3 × 102, 2.5 × 10–1 to 6 × 100,and 9 × 10–2 to 1.5 × 100 m2/day for chickpea, pea, and vetch seeds, respec-tively. The lower values were for low seed coat hydration and increasedwith increasing coat hydration. Seed coat diffusivities to water, being muchlower than those of a whole seed, may be considered to restrict imbibition.However, a decrease in water content, soil water conductivity, or seed-soilcontact area, combined with low seed coat diffusivity to water, may restrictseed imbibition to a great extent (Dasberg and Mendel, 1971; Hadas, 1970;Hadas and Russo, 1974a; Williams and Shaykewich, 1971; Ward andShaykewich, 1972).

Seed-Soil Interface Geometrical Configuration

The geometrical configuration of the seed-soil interface zone depends ona combination of seed coat surface properties, seed dimensions, and soilstructure around the seed (Koller and Hadas, 1982; Hadas, 1982). Seedsplaced on the soil surface or buried in a moist soil have partial contact withthe soil particles; these contact points are few and of small area, thus theirnumber and total area become negligible for seeds lying on the soil surface.The smaller the soil units are relative to the seeds, the greater the number ofcontact points and the total contact area will be (Hadas and Russo, 1974b;Hadas, Wolf, and Meirson, 1978). When these contact points are wettedwith water, the contact area increases because water films and water collarsform around the contact points; their shape and dimensions depend on therelative sizes of the seeds and the soil particles and on the water content(Collis-George and Hector, 1966; Hadas and Russo, 1974a,b). If the seedsare coated with a hairy cover, contact will be minimal unless the hairy coveris removed or the soil is compacted around the seeds. The wetted contact ar-eas may be contiguous with impermeable sections of the seed coat, render-ing these areas ineffective in transporting water to the imbibing seed. Some-times, a minute contact point that touches a permeable area, identified as aport of entry (e.g., chalza, micropyle), can adequately supply the waterneeded by the imbibing seed (Berggren, 1963; Hyde, 1954; Manohar andHydecker, 1974; Spurny, 1973). Most seeds, other than those sown in agri-cultural areas, once dispersed come to rest on the soil surface or fall intocracks, and their germination depends on: (1) enhanced seed-soil contactarea because of soil surface roughness (Winkle, Roundy, and Box, 1991);

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(2) wet priming (Finch-Savage and Pill, 1990); (3) swollen mucilaginousseed coats (Koller and Hadas, 1982); or (4) specific built-in burial mecha-nism (Gutterman, Witztum, and Evenari, 1967; Koller and Hadas, 1982;Meyer and Poljakoff-Mayber, 1989; Young and Evans, 1975).

Impedance to Water Flow Across the Seed-Soil Contact Zone

Under natural conditions, the hydraulic properties of the seed-soil con-tact zone vary during imbibition because of changing soil water content andseed-soil contact area. Moreover, these variations cannot be directly mea-sured. Trials aimed at determining the effects of soil and soil water conduc-tivity on seed water uptake and germination have been carried out in poroussubstrates, soil plugs on sintered glass, or other materials. Either water flowwas found to be restricted to a segment of the seed surface or the seeds weremechanically confined so that their swelling and imbibition were inhibited(Collis-George and Sands, 1959, 1962; Collis-George and Hector, 1966;Dasberg, 1971). The data interpretation in these studies was criticized byvarious researchers, who pointed out that the experimental procedures ledto the observation of the combined effects of soil water content, soil me-chanical stresses, water conductance, and seed-soil interface on germina-tion (Hadas, 1970; Hadas and Russo, 1974a; Sedgley, 1963). Hadas andRusso (1974a,b) developed and used a procedure to enable determination ofthe separate effects of each of the water potential components, capillaryconductivity to water, and seed-soil contact on seed germination. Their ex-perimental results led to the conclusion that seed-soil contact impedance toflow increases with the decreasing seed wetted area, soil conductivity towater, or both. Contact impedance to water flow for a given size of seed andfor a given m increases with increasing coarseness of the soil texture,structure, or both. The final germination percentage was not affected by ei-ther soil or K( w), as long as soil was higher than b (Hadas and Russo,1974a,b).

The model derived by Hadas and Russo (1974b) furnished a correlationbetween seed-soil water contact impedance and either wetted percentage ofseed surface area or K( w). Solutions of Equation 1.2, with the appropriateboundary and initial conditions for each part of the soil-seed system, en-abled prediction of water uptake time courses that agreed well with data ob-tained in the laboratory and in small field plots. Those preliminary predic-tion capabilities prompted Hadas (1977a) to attempt to extend the model bycorrelating the predicted or measured imbibition time with the transitionphase duration and final germination percentage. Good agreement was at-tained by using the extended model to predict the final germination and

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compare the forecast values with observed ones. These models, which havea physical basis, were found to be too cumbersome and unsuitable for prac-tical application and their further refinement was abandoned.

These models, although not practical themselves, suggest some practicalapplications, namely that proper seedbed preparation which decreases theproportion of large-sized soil crumbs in the seedbed can control potentialdecreases in water uptake, rate of germination, and final germination per-centage (Currie, 1973; Hadas, Wolf, and Meirson, 1978). Many seeds swellduring imbibition, and the swollen seed imposes compacting forces on theparticles around it, thus improving its contact with the soil and reducingseed-soil contact impedance. Concurrently, water uptake reduces soil watercontent next to the seed surface, while mechanical constraints and imped-ance to flow may increase. The combined effect of the two contrary trendsmay cause: (1) no change in the seed-soil interface impedance and, there-fore, no delay of germination (Hadas, 1970, 1977b); (2) the effects of reduc-tion in water content and water conductance to be greater than the effects ofreduction in seed-soil contact impedance, thus impairing germination; or(3) the effects of reduced impedance to flow to be greater than those of re-duced water content and conductance, so that water flow to the seed will notbe restricted. It is obvious that the swelling of seeds lying on the soil surfacewill not compact the soil underneath, but rather will reduce their contactarea, so that increased impedance to flow, reduced water uptake, and de-layed germination are to be expected.

Under saline conditions, observed contact impedance effects may bepartially obscured by the accumulation of excluded salts at the seed surface,which will reduce water uptake, germination rate, and final germinationpercentage, even though no changes in impedance to flow occurred (Hadas,1970, 1976; Williams and Shaykewich, 1971).

The current knowledge of seed behavior during germination and their re-sponses to changes in the soil environment has improved our understandingof the required conditions and properties of a seedbed. Although the variousapproaches to forecasting seed water uptake are, at best, good approxima-tions, they do provide methods to be followed when planning seedbed prep-arations (Hadas and Russo, 1974a,b). It is evident that the crucial factors inseedbed preparation are control of the seed-soil contact area and the imped-ance to flow across the interface. This statement is based on the observa-tions cited in the previous paragraphs. However, to date, the results of directmeasurements of soil environmental conditions and of germination behav-ior have been either inconsistent or incomplete; therefore, we are left withlarge knowledge gaps which can be attributed to many factors. Probably themost important factors are (1) the extreme complexity of the soil system, in-cluding soil structure, stability, and hydraulic properties, and (2) theoretical

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aspects and experimental difficulties in the microscale analysis of flowaround and into a seed and across the seed-soil interface.

Modeling Seedbed Structure: Temporal and Spatial Evolutionof Seedbed Physical Properties

Any modeling effort aimed at characterizing seedbed physical propertiesand configuration has to rely on several criteria of soil structure characteris-tics and their spatial and temporal variations from initial preparation untilseedling establishment. Such an endeavor first requires analytical presenta-tion or modeling of the soil structure architecture, the physical propertiesderived from that architecture, the temporal and spatial evolution of soilstructure changes, and the concurrent changes in the physical properties.Moreover, those procedures must be applied at various scales: soil structurestability must be considered at aggregate or subaggregate size, seed size,and on larger scales for a field stand (Hadas, 1997; Guerif et al., 2001).

In a detailed review Letey (1991) stated that soil structure does not lenditself to quantification. His statement was based on his recognition of thecomplexity involved in quantifying the heterogeneous soil structure. Tre-mendous knowledge gaps exist between what can be technically or experi-mentally obtained and the information and kind of data required for theoret-ical analysis. Young, Crawford, Rappoldt (2001), following Dexter (1988),who stated that spatial heterogeneity = spatial variability = soil structure,came to the conclusion that an explicit account of the heterogeneity inher-ent in the soil physical architecture has until recently been beyond experi-mental and theoretical insight.

These observations on the current state of the art indicate that special ef-forts are required to extend, improve, and create experimentally obtaineddatabases which will yield empirical relationships between aggregate size,water regime, and structural stability. From these relationships, estimates ofphysical properties could be derived by means of currently existing models,operated at minute time and space steps. Taking heat, water, and air to be themajor environmental factors involved in seed germination forecasting, themodels chosen must be based on physical laws and must combine mass andenergy fluxes and conservation principles. Moreover, although the numeri-cal procedures will be complex, hard to follow, and difficult to handle, thesemodels need to be validated. Partial efforts have already been made. Guptaand colleagues (1991) examined models used for predicting soil bulk den-sity, water retention and conductivity, thermal conductivity, heat capacity,and gaseous diffusion with respect to their adaptation to fractured and com-pacted soils, and their critical examination identified shortcomings of the

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models. They pointed out knowledge gaps that require further research andindicated the difficulties to be expected in closing these gaps. Models of si-multaneous heat and water transport have been developed for homogeneoussoils (Nasser and Horton, 1992; Mullins et al., 1996), mulched soils or two-layered soils (Bristoe and Campbell, 1986), heterogeneous soils (Chungand Horton, 1987; Hares and Novak, 1992), and for ridged seedbeds(Benjamin, Ghaffarzadech, and Cruse, 1990; Gupta et al., 1990) and gas-eous exchange (Richard and Guerif, 1988a,b). However, these models willhave to be modified to include temporal changes in soil structure and the re-sulting variations in the physical properties.

Modeling soil fragmentation on the basis of soil dynamics, classical soilmechanics, and critical state theory, in an effort to predict the final soilstructure, is only partly possible (Hettariachi, 1988), yet none of these mod-els nor their extensions enable prediction of soil structure and seedbed tilth(Hadas, Larson, and Allmaras, 1988; Hadas, 1997). This statement is stillvalid (Guerif et al., 2001). These observations show that modeling of soilfragmentation is still a rather remote goal whose attainment will require tre-mendous efforts and much time.

CONCLUDING REMARKS

Timely, fast, and uniform seed germination, emergence, and final standattainment are crucial for a successful crop and high yields, yet many fieldstudies lack crucial information. Seeds deposited or sown respond individu-ally to the microenvironment surrounding them. To specify the favorablesoil physical properties, chemical constituents, microbiological populationactivity, and their interactions with one another and the climate would be aninsurmountable task, in light of our current knowledge. Moreover, themicroenvironment to which a seed responds tends to vary greatly and toinduce great spatial variability across the field. Agricultural experience sug-gests that the soil physical properties are the major determinants of a suc-cessful seedbed conducive to optimal seed germination and stand attain-ment.

Although each seed reacts individually to its microenvironment, a fieldconsists of a wide range of microenvironments. Since stand establishmentunder field conditions is our task, our approach to achieving that involvesunderstanding how seeds germinate under field conditions. When cereals orgrasses are considered, tillering is expected to correct the adverse effects ofa poor stand, but for other crops, reduced emergence and low stand unifor-mity are associated with poor seedbed preparation (Perry, 1973; Hadas,Wolf, and Rawitz, 1983; Hadas and Wolf, 1984; Hadas et al., 1990).

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There are great difficulties in tailoring recommended seedbeds, sincefield conditions present a great variety of soil structure stability, climaticuncertainties, and traffic history, all of which affect the performance of thenext crop. Field conditions are difficult to reproduce under laboratory con-ditions, and, until recently, attempts to correlate laboratory studies (com-plex as they may be) failed to create a reliable database for field perfor-mance predictions. The material presented and discussed in the previouschapters presents a small variety of studies carried out to resolve that com-plex system and to furnish a reliable methodology for specifying the desiredseedbed and the means to produce it. It is obvious that greater effort should bedirected toward both the basic understanding of seed germination and thesearch for proven methodologies for specifying the proper seedbeds andrecommending the means to achieve them.

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Hadas, A. (1976). Water uptake and germination of leguminous seeds under chang-ing external water potential conditions in osmotic solutions. Journal of Experi-mental Botany 27: 480-489.

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Hadas, A., Shmulewich, I., Hadas, O., and Wolf, D. (1990). Forage wheat yields asaffected by compaction and conventional vs. wide frame tractor traffic patterns.Transactions of the ASAE 33: 79-85.

Hadas, A. and Stibbe, E. (1973). Analysis of water uptake and growth patterns ofseedlings of four species prior to emergence. In Hadas, A., Swartzendruber, D.,Rijtema, P.E., Fuchs, M., and Yaron, B. (Eds.), Physical Aspects of Soil Waterand Salts in Ecosystems (97-106). Ecological Studies 4. Berlin: Springer Verlag.

Hadas, A. and Stibbe, E. (1977). Soil crusting and emergence of wheat seedlings.Agronomy Journal 69: 547-550.

Hadas, A. and Wolf, D. (1983). Energy efficiency in tilling dry clod forming soils.Soil Tillage Research 3: 47-59.

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Hadas, A. and Wolf, D. (1984). Refinement and reevaluation of the drop shatter soilfragmentation method. Soil Tillage Research 4: 237-249.

Hadas, A., Wolf, D., and Meirson, I. (1978). Tillage implements-soil structural rela-tionships and their effects on crop stands. Soil Science Society of America Jour-nal 42: 632-637.

Hadas, A., Wolf, D., and Rawitz, E. (1983). Zoning soil compaction and cottonstand under controlled traffic conditions. Paper No. 83-1042. ASAE 1983 Meet-ing, Bozeman, Montana.

Hadas, A., Wolf, D., and Rawitz, E. (1985). Residual compaction effects on cottonstand and yields. Transactions of the ASAE 28: 691-696.

Hadas, A., Wolf, D., and Stibbe, E. (1981). Tillage practices and crop response an-alysis of agro-ecosystems. Agro-Ecosystems 6: 235-248.

Hanks, R.J. and Thorp, F.C. (1957). Seedling emergence of wheat, grain sorghumand soy-beans as influenced by soil crust strength and moisture content. Soil Sci-ence Society of America Proceedings 21: 357-360.

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Hegley, G.H. (1995). Seed germination in the soil: Dormancy cycles. In Kigel, J.and Galili, G. (Eds.), Seed Development and Germination (pp. 529-541). NewYork: Marcel Dekker.

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Holmes, J.W., Greacen, E.L., and Gurr, C.G. (1960). The evaporation of water frombare soils with different tilths. Transactions of the Seventh International Con-gress of Soil Science 1: 188-194.

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Jackson, R.D. (1973). Diurnal changes in the soil water content during drying. InBruce, R.R, Flach, K., and Taylor, H.M. (Eds.), Field Soil Water Regime (pp. 37-55). Special Publication #5. Madison, WI: Soil Science Society of America.

Karssen, C.M. (1970).The light promoted germination of the seeds of Chenopodiumalbum L.: V. Dark reactions regulating quantity and the rate of response to redlight. Acta Botanica Neerlandica 19: 187-196.

Kebreab, E. and Murdoch, A.J. (1999a). A model of the effects of a wide range ofconstant and alternating temperatures on seed germination of four Orobanchespecies. Annals of Botany 84: 549-557.

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Marshall, T.J., Holmes, J.W., and Rose, C.W. (1996). Soil Physics. Cambridge,United Kingdom: Cambridge University Press.

Martin, V.L., McCoy, E.L., and Dick, W.A. (1990). Allelopathy of crop residues in-fluences corn seed germination and early growth. Agronomy Journal 82: 555-560.

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Mullins, C.E., Townend, J., Mtakwa, P.W., Payne, C.A., Cowen, G., Simmonds,L.P., Daamen, C.C., Dunbabin,T., and Naylor, R.E.L. (1996). Emergence UserGuide: A Model to Predict Crop Emergence in the Semi-Arid Tropics. Aberdeen,United Kingdom: Department of Plant and Soil Science, Aberdeen University.

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Perfect, E., Zhai, Q., and Belvins, R.L. (1997). Soil and tillage effects on the charac-teristic size and shape of aggregates. Soil Science Society of America Journal 61:1459-1465.

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Richard, G. and Guerif, J. (1988b). Modelisation des transferts gazeux dans le litsemence: Application au diagnostic des conditions d’hypoxie des semeneces debeterave sucriere (Beta vulgaris L.) pendent la germination. II: Resultats dessimulations. Agronomie 8: 639-646.

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Ward, J. and Shaykewich, C.F. (1972). Water absorption by wheat seeds as influ-enced by hydraulic properties of the soil. Canadian Journal of Soil Science 52:99-105.

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Chapter 2

Models to Describe and Predict the Effects of Seedbed EnvironmentThe Use of Population-Based Threshold Modelsto Describe and Predict the Effects of Seedbed

Environment on Germination and SeedlingEmergence of Crops

William E. Finch-Savage

INTRODUCTION

Importance of Seedling Emergence to Crop Production

Seed germination and subsequent seedling growth to emergence fromthe soil are crucial steps in crop production. Although some field crops suchas cereals can compensate for low stands by tillering, in many crop speciesno amount of effort and cost during plant growth can compensate for poorseedling establishment. A wide range of biotic and environmental factorsinteract with the potential performance of the seed lot to determine the suc-cess of seedling establishment (Hegarty, 1984). This chapter will use popu-lation-based threshold models to summarize current understanding of theinteraction between the seedbed environment and the seed population fromsowing to seedling emergence. The potential for these threshold models topredict seedling emergence in the field will then be discussed while de-scribing the construction of an example simulation. In order to see the rele-vance and importance of the studies reviewed, it is necessary to briefly out-line the consequences of nonoptimal seedling emergence in crops.

The timing, pattern, and extent of seedling emergence have a profoundimpact on crop yield and market value (Finch-Savage, 1995). Only part of

I would like to thank my colleagues Hugh Rowse, Kath Phelps, and Richard Whalleywith whom I have collaborated over a number of years on crop establishment projects, inparticular Hugh Rowse for use of his unpublished work. I thank the U.K. Department forEnvironment, Food and Rural Affairs (DEFRA) and, more recently, the Department forInternational Development (DFID) who have funded these collaborations.

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the total biomass produced is harvested, and this component is crop andmarket specific. This economic yield is often determined by the whole plantpopulation as a bulk weight per unit area, as in grain or sugar beet crops; inmany horticultural crops economic yield is determined by individual plantswithin the population, for example, the number of plants within closely de-fined size grades (e.g., carrots, onions) or the number of plants that “ma-ture” at a single harvest (e.g., lettuce). The effects of seedling emergence oneconomic yield are generalized in Figure 2.1. As the number of seedlings

Tota

l yie

ldP

lant

size

Num

ber

Plant density

a)

b)

Plant density

Required size grades

Requiredsize grades

Plant size

c)

FIGURE 2.1. Schematic illustration of the effects of seedling emergence on mar-ketable yield. Total yield increases asymptotically with increasing plant density(a) while there is a concurrent decrease in the size of individual plants (b). Theuniformity of plant size at harvest determines the proportion of the crop in therequired size grades (c, - - - - nonuniform crop, ______ uniform crop).

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emerging per unit area (crop density) increases, yield increases asymptoti-cally (Figure 2.1a), but the size of individual plants decreases (Figure 2.1b).Thus target populations need to be achieved to grow bulk crops cost-effec-tively and to produce plants of the appropriate sizes for graded yields in hor-ticultural markets. Crop density can also influence time taken to reach ma-turity (e.g., onions, Mondal et al., 1986) and the uniformity of plants atmaturity (e.g., cauliflower, Salter and James, 1975). It is also commonplaceto oversow crops to avoid a limiting density, and this, under favorable con-ditions, results in densities that are too high. This situation can producepoor canopy structure which delays and reduces the uniformity of maturity.

Variation in the time to seedling emergence within the population can ac-count for much of the subsequent variation in plant size during crop growthto harvest (Benjamin and Hardwick, 1986; Benjamin 1990). The ranking ofseedling size at the end of emergence changes little with time, and in manycases the difference between plants increases during growth (Benjamin andHardwick, 1986). Thus more uniform seedling emergence can result in agreater proportion of the population falling within the required high-valuesize grade or maturation period to increase crop value (Figure 2.1c). Rapidand predictable emergence following sowing is particularly important in re-gions where season length limits yield (e.g., grain maize, Breeze andMilbourne, 1981; sweet corn, Cal and Obendorf, 1972), where water re-sources for irrigation are limited (Jordan, 1983), or when crops are grown ina programmed sequence of sowings (e.g., lettuce, Gray, 1976). Rapid emer-gence can also increase crop competitiveness with weeds emerging fromthe soil seedbank and facilitate earlier application of herbicides whenweeds are more susceptible. Seedling emergence has an impact on manyother aspects of crop management and its cost-effectiveness, not least be-cause many of the costs of production (e.g., fertilizers and disease and pestcontrol) are likely to be similarly independent of the success of seedlingemergence.

The time between sowing and seedling emergence can be convenientlydivided into the phase before and after germination. Although these effectsare confounded in most seedling-emergence studies, the two phases areeach uniquely affected by adverse seedbed conditions (Finch-Savage, 1995).It is thought that the timing of germination can account for much of the vari-ation in seedling emergence time (Finch-Savage and Phelps, 1993; Finch-Savage, Steckel, and Phelps, 1998), whereas seedling losses and variationin the spread of seedling emergence times within the population occurlargely in the postgermination growth phase (Hegarty and Royle, 1978;Durrant, 1981; Finch-Savage, Steckel, and Phelps, 1998). Therefore, to pre-dict the impact of seedbed environment on seedling emergence both phasesmust be considered.

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Seedbed Environment

The seedbed environment provides a highly variable and often hostileenvironment for seedling emergence from crop seeds and those in the weedseed bank. For germination, most crop seeds require water, adequate tem-perature, and a favorable gaseous environment. Dormancy has little impacton seedling emergence of most commercial crops (Villiers, 1972; Maguire,1984) but is a major factor in the emergence of weeds (Baskin and Baskin,1998). For the weed seeds there are additional germination-promoting fac-tors such as light and nitrate to consider (Hilhorst and Karssen, 2000).Modeling nondormant crop seed germination is therefore less complex andfurther aided because the crop seed is generally the same age and is sown ata narrow range of depths into the soil, so the environment for germination ismore uniform within the population.

Crop seeds are sown close to the surface, and therefore soil water contentand temperature can vary widely. Reduced oxygen availability can alsohave a major impact on germination and seedling emergence (Corbineauand Côme, 1995). This occurs when there is excessive water in the seedbed(e.g., Dasberg and Mendel, 1971; Hegarty and Perry, 1974; Perry, 1984), orwhen a soil crust forms to seal the seedbed surface or engulf the seed (Rich-ard and Guérif, 1988a,b). Sensitivity to oxygen partial pressure (pO2) dif-fers among species, and linear relationships have been shown between ger-mination rate and the logarithm of pO2 (Al-Ani et al., 1985). This suggeststhat a threshold model, as discussed in the following for temperature andwater potential, could be applied to this relationship. However, followinggood seedbed preparation, the oxygen concentration in the soil atmospherein most cases does not fall below 19 percent (Richard and Boiffin, 1990).Crust formation and limiting oxygen environments occur only intermit-tently, and their effect can be minimized by good seedbed preparation(Chapters 1 and 3), whereas the variable strength of soil through whichseedlings grow after germination is always a factor. The remainder of thischapter will be concerned with the effects of the three ubiquitous seedbedfactors, water availability, temperature, and soil strength, that largely deter-mine the patterns of germination and seedling emergence of crops observedin the field.

IMBIBITION

Water uptake by the seed generally occurs in three phases: rapid initialuptake, a lag phase with limited further uptake, and then a second phase ofrapid water uptake associated with radicle emergence (Bewley and Black,

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1994). Imbibition is identified with the first phase of water uptake and is re-garded as a physical process, although metabolism is initiated before seedsreach full moisture content. Initial water uptake is driven by matric forcesresulting from the hydration of cell walls, starch and protein bodies, etc. Asthe physiological range of water contents is approached there is a greaterdependence on osmotic potential determined by the concentration of dis-solved solutes. The rate of early water uptake can have a large negative im-pact on seed viability and the success of seedling emergence. If imbibitionis too rapid, damage may be caused both directly and through a positive re-lationship with chilling injury. The extent of this damage is directly relatedto the integrity of the seed coat and other aspects of seed vigor (reviewed byWoodstock, 1988; Vertucci, 1989; Finch-Savage, 1995).

Imbibition can have an important influence on the prediction of germina-tion and emergence times when seeds are sown into dry soils or when thecontact between seed and soil is poor and therefore also likely to be variablein the seed population. The seed coat and other tissues can also have an im-portant regulating affect on water uptake (e.g., soybean, McDonald, Ver-tucci, and Roos, 1988a) by controlling permeability. The movement of wa-ter into the seed is driven by gradients of water potential between the seedand the surrounding soil. Mechanistic models of imbibition have also beendeveloped based on water concentration (diffusivity theory) rather than wa-ter potential gradients (hydraulic conductivity theory). This is a convenientsimplification that can be used in homogeneous environments. When con-sidered as a whole, the flow of water through the soil and into the seed is nota homogeneous system and is therefore considered here in terms of hydrau-lic flow. In this case, the rate of water uptake, in simple terms, is governedby the hydraulic conductivity of the seed and the soil and driven by the wa-ter potential gradient between them.

A reduction in water potential of the surrounding soil will therefore re-duce the rate of water uptake by the seed because the gradient between themis less. However, the effect on rate is not directly proportional to changes inthe gradient as hydraulic conductivity is also altered. Hydraulic conductiv-ity is a function of the permeability of the seed and surrounding soil, the ex-tent of contact between them, and temperature (reviewed by Bewley andBlack, 1978; Vertucci, 1989). For example, rate of water uptake increaseswith temperature (Vertucci and Leopold, 1983). The situation is furthercomplicated because there appears to be a wetting phase before hydraulicflow is initiated and hydraulic conductivity changes as the seed swells dur-ing imbibition (Vertucci, 1989). Soil water potential gradients may alsoform at the interface with the seed, and the relative importance of vaportransport of water to seed may be underestimated in many studies (Wuest,Albrecht, and Skirvin, 1999). In addition, seed coatings that are now com-

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monly used in agriculture also influence imbibition (Schneider and Renault,1997). Therefore, perhaps inevitably, seed imbibition under variable seed-bed conditions is complex. Nevertheless, a number of models have been de-veloped that make a range of different assumptions, and these have been re-viewed in detail elsewhere (Hadas, 1970, 1982; Dasberg, 1971; Bruckler,1983a,b; Bouaziz and Bruckler, 1989a,b; Vertucci, 1989; Schneider andRenault, 1997; Chapter 1).

Additional points to consider are that the different chemical composi-tions of seeds will affect the amount of water they take up; for example,equilibrium moisture content at any given water potential will always begreater in pea than soybean (Vertucci and Leopold, 1987). Equilibriummoisture contents also differ among seed tissues, often with the embryonicaxis having a higher water content than the storage tissues (e.g., soybean,McDonald, Vertucci, and Roos, 1988b; maize, McDonald, Sulivan, andLauer, 1994).

GERMINATION

The initiation of radicle growth at the end of the lag phase of imbibitionterminates germination sensu stricto, and therefore germination is generallyrecorded when radicle growth is first observed. Following germination,desiccation tolerance is lost progressively during growth of the radicle, inmost species, and so the initiation of growth is a critical step in the progres-sion from sowing to seedling emergence. This critical step will occur at dif-ferent times in each seed within the population, leading to a distribution ofgermination times and the characteristic sigmoidal cumulative germinationcurve. In agriculture, this spread of germination times can be very undesir-able for the reasons described, but under natural conditions it presents agood strategy to cope with the highly variable conditions of temperatureand water potential in the surface of the soil where seeds germinate. In theabsence of significant disease, the interaction of this characteristic seedlotdistribution of germination times with soil temperature and water potentiallargely determines the timing of seedling emergence in crops. Understand-ing this interaction and developing ways to model the outcome is essentialto developing effective crop establishment practices. Population-basedthreshold models provide a useful framework for this purpose. Within thesemodels the rate of development, such as progress toward germination orseedling growth, increases above a base (threshold) value for a given factor(temperature, water potential, hormone concentration, etc.). Below the basevalue, development ceases. The effect of the factor on rate of developmentabove the base is described by an appropriate mathematical function. In

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many cases this function is linear. The base values may differ among indi-viduals in the population and are therefore important in describing differ-ences in their response to the factor concerned. The bases that are likely tohave physiological importance (Welbaum et al., 1998; Meyer, Debaene-Gill, and Allen, 2000; Bradford, 2002) can be determined either explicitlyby measuring the value at which development ceases or estimated implic-itly in the case of linear relationships by extrapolation of the fitted line tothe intercept.

Threshold Models: Effects of Temperature and Water Potential

For the purpose of modeling germination of nondormant seeds it is gen-erally assumed that seeds germinate in a set order and that this order is notaffected by germination conditions. Each seed can therefore be assigned avalue of G, which is the fraction of the population at which it germinates(e.g., G10, G50, and G90 in Figure 2.2). The percentage of seeds that will ger-minate as well as germination time and spread of times within the seed pop-ulation are all greatly influenced by temperature (reviewed by Roberts,1988; Probert, 2000) and water potential (reviewed by Bradford, 1990,1995, 2002).

Temperature

Seeds can germinate over a wide range of temperatures, but maximumpercentage germination is typically reduced at the extremes of the range(Labouriau and Osborn, 1984; Roberts, 1988; Probert, 2000). Individualseeds within the population can therefore have different levels of tolerance(thresholds) at both high and low temperatures. For any individual seed inthe population, germination rate, which is the reciprocal of germinationtime, increases from a base to an optimum temperature above which it de-creases to a ceiling temperature that indicates the limit of its tolerance(Labouriau, 1970). In many cases this response to temperature can be de-scribed by linear relationships where the base and ceiling temperatures aredefined by the intercepts on the temperature axis where rate tends to zero(Figure 2.2a, Labouriau, 1970; Bierhuizen and Feddes, 1973; Garcia-Huidobro, Monteith, and Squire, 1982a). A linear relationship at sub-optimal temperatures has been shown for a wide range of species, for exam-ple, many temperate vegetables (Wagenvoort and Bierhuizen, 1977), otherherbaceous species (Steinmaus, Prather, and Holt, 2000; Trudgill, Squire,and Thompson, 2000), subtropical crops (Covell et al., 1986), range grasses,and shrubs (Jordan and Haferkamp, 1989). At suboptimal temperatures a

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heat sum (Feddes, 1972; Bierhuizen and Feddes, 1973; Bierhuizen andWagenvoort, 1974), more recently called thermal time (Garcia-Huidobro,Monteith, and Squire, 1982a), approach can therefore be used to predictgermination time:

T1(G) = [T-Tb(G)] t(G) (2.1)

where, T1(G) is the thermal time to germination of percentile G, T is thetemperature, Tb(G) is the base temperature, and t(G) is the time taken forgermination of that percentile. In this context thermal times have been used

50%

Topts

c)

YbTc

b)a)

Tb

1/qT

Ger

min

atio

n ra

te[1

/]

t (G

)

Ger

min

atio

n ra

te[1

/]

t (G

)

Temperature( C)T, °

Water potential( , MPa)Y

FIGURE 2.2. Schematic illustration of the effects of temperature (a) and waterpotential at suboptimal temperature (b) on the rate of germination. G10 (dottedlines), G50 (dashed lines), and G90 (solid lines) represent individual seeds in thepopulation at percentiles 10, 50, and 90, respectively. Germination rate in-creases linearly with temperature above a base (Tb). The slopes of these linesare the reciprocal of the thermal times to germination (1/ T). As temperature in-creases above an optimum (Topt), rate of germination decreases to a ceilingtemperature (Tc). Rate of germination also decreases linearly with water poten-tial ( ) to a base ( b). Tb is common to all seeds in the population, but 1/ T , Tc,and b vary among seeds in a normal distribution (c). For further explanation ofthese parameters see the text.

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most often to compare and predict time to 50 percent germination (G50). Inmany cases little variation in Tb(G) has been shown among individual seedswithin the population, and Tb is therefore considered to be a constant al-though Tb differs greatly between species (Covell et al., 1986). Therefore,thermal time to germination of a given percentile is constant, but each per-centile requires different thermal times to complete germination. Germina-tion rate 1/t(G) is linearly related to temperature, but with a different slopefor each percentile (e.g., G10, G50, and G90 in Figure 2.2a):

1/t(G) = (T-Tb)/ T1 (G) (2.2)

At supraoptimal temperatures the rate of germination declines in a series ofparallel lines (Figure 2.2a). The intercepts therefore differ in the populationand thus:

1/t(G) = [Tc (G) – T]/ T2 (2.3)

where Tc (G) is the ceiling temperature and T2 is thermal time. A naturalextension of the thermal time approach allows the description of germina-tion times of the whole population at a range of suboptimal temperatures byassuming Tb is constant and using a distribution to describe variation in T1(G) (Covell et al., 1986; Ellis et al., 1986; Ellis, Simon, and Covell, 1987). Ifvariation in T1 (G) is normally distributed (Figure 2.2c) then probits can beused:

1/t(G) = (T-Tb)/ {[probit (G) – K] } (2.4)

where is the standard deviation of T1 (G) and K is a constant. Atsupraoptimal temperatures T2 remains constant and variation in germina-tion rate is accounted for by a normal distribution of Tc (G) (Ellis et al.,1986; Ellis, Simon, and Covell, 1987) so that

1/t(G) = ({[Ks – probit (G)] } – T)/ T2 (2.5)

where is the standard deviation of Tc (G) and Ks is a constant. Other distri-bution functions may be more appropriate to describe variation in thermaltime for other species (e.g., Washitani, 1985; Covell et al., 1986; Ellis andButcher, 1988). Equations 2.4 and 2.5 can be used to predict germinationrate at any constant temperature for all seeds in the population.

In this work Ellis and colleagues used repeated probit regression analy-ses (Finney, 1971) of germination data from all the temperatures recordedto determine the best fit (least residual variance) to the data. Bradford

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(1995) points out that a requirement of probit analysis is that samples ateach time point should be independent (Finney, 1971). However, in usualpractice, repeated measurements are made from a single sample to deter-mine cumulative germination curves, rather than single measurements froma number of samples. Thus data do not conform to the criterion of independ-ence. Bradford (1995) argues that for practical purposes, the results of thetwo methods are identical (Campbell and Sorensen, 1979). He continues,that although statistical comparisons based on probit analysis from cumula-tive scored data are invalid, other procedures developed specifically for thecumulative curve are available (Bliss, 1967). This same caveat applieswhere the use of probit analysis is mentioned in the following.

Water Potential

As with temperature, the rate of progress toward 50 percent germinationhas been shown to be linearly related to water potential (Hegarty, 1976).Gummerson (1986) was the first to consider germination in hydrotime, ascale analogous to thermal time that can be used to describe the response ofseeds to different water potentials. The wider relevance of this concept wasrealized by Bradford who then developed and extended the use of hydro-time to provide insight into a wide range of seed behavior. A full review ofthe use of hydrotime is beyond the scope of this work but has been elo-quently covered in detail elsewhere by Bradford and colleagues (Bradford,Dahal, and Ni, 1993; Bradford, 1995, 2002). Here, hydrotime will be con-sidered only in relation to its contribution to the prediction of germinationtimes for agricultural purposes.

The hydrotime ( H) approach considers germination rate as a function ofthe extent to which seed water potential exceeds a base water potential be-low which germination will not occur. It is analogous to thermal time andthus:

H = [ – b(G)] t(G) (2.6)

where b(G) is the base below which germination of percentile G willnot occur. If H is a constant, then the time required for germination [t(G)]of percentile G is inversely proportional to the amount by which seed ex-ceeds its base water potential [ b(G)]. By analogy to Equation 2.2:

1/t(G) = [ – b(G)] / H (2.7)

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Figure 2.2b shows that unlike Tb, but like Tc, b is thought to differamong individual seeds in the population, and these differences result in thedifference in the time seeds take to germinate (Gummerson, 1986; Brad-ford, 1990; Dahal and Bradford, 1990). b is negatively related to germina-tion rate, so slow-germinating seeds have the highest b. In this case the dif-ference between seed and b(G) is least, so hydrotime accumulates moreslowly (Figure 2.3). With H constant, differences in germination rate aretherefore determined solely by the variation in b that approximates to anormal distribution (Gummerson, 1986; Bradford, 1990; Dahal and Brad-ford, 1990). As seed decreases – b decreases and therefore hydrotimeaccumulates more slowly and the whole population of seeds take longer togerminate in clock time. When seed is reduced to less than its b then itwill not germinate. Therefore in experiments with fixed water potentialswithin the range of b not all seeds will germinate. However, although the

Ger

min

atio

n%

Days from sowing

100

80

60

40

20

05 10 15 20 250

Tb b= 2.1°C, = –0.8 MPa

Tb b= 2.1°C, = –0.9 MPa

Tb b= 2.1°C, = –1.0 MPa

HT = 47 MPa °Cd

FIGURE 2.3. The influence of b(G) on the shape of the cumulative germinationcurve at suboptimal temperatures in carrot. Values of Tb and b are shown forpercentiles G25, G50, and G75. According to the basic hydrothermal time con-cept, all seeds have the same Tb, but more rapidly germinating seeds have alower b(G) and so – b(G) is greater and therefore more hydrothermal time( HT) is accumulated per unit of clock time. As HT to germination is the same forall seeds in the population (47 MPa °Cd), seeds with a lower b will germinatefirst when their accumulated HT = 47 MPa °Cd. The shape of the cumulativegermination curve is therefore determined by the distribution of b(G). (Source:Data from Finch-Savage, Steckel, and Phelps, 1998.)

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seed will not complete germination and initiate radicle growth below b,metabolism continues, and this has consequences for the timing of germi-nation and therefore its prediction as discussed later in this chapter (sectionSeed Advancement Below Base Water Potential).

The repeated probit analysis technique used by Ellis and colleagues(1986) for thermal time was adapted by Bradford (1990) to describe the af-fect of water potential on germination for the whole population of seeds:

Probit (G) = { – [ H / t(G)] – b(50)} / b (2.8)

where b(50) is the median b and b is the standard deviation of bamong seeds in the population. Following this analysis time courses of ger-mination at a range of water potentials can be mapped onto a commonhydrotime scale (Bradford, 1990, 1995; Dahal and Bradford, 1990; Ni andBradford, 1992, 1993; Bradford and Somasco, 1994).

Water Potential and Temperature

Gummerson (1986) developed a combined description of the response ofseeds to temperature and water potential in the theory of hydrothermal time.According to this theory, rates in thermal time are proportional to water po-tential and can therefore be described by an equation similar in form toEquation 2.2:

1/ T(G – b(G HT (2.9)

where HT (hydrothermal time) is a constant. Gummerson (1986) combinedEquations 2.2 and 2.9 to give:

HT = [ – b(G)] (T – Tb) t(G) (2.10)

Consistent with the development of this theory, HT and Tb are assumedconstant and b varies with (G). As pointed out by Gummerson (1986), it ispossible that these assumptions are not entirely correct and this will be dis-cussed further in the section Further Development of Threshold Models.Nevertheless, this approach has been shown to adequately describe germi-nation curves produced in a wide range of combinations of constant temper-ature and water potential (Gummerson, 1986; Dahal and Bradford, 1994;Finch-Savage, Steckel, and Phelps, 1998; Roman et al., 1999; Shresthaet al., 1999; Allen, Meyer, and Khan, 2000). Accepting these assumptions,it is possible to describe the effect of suboptimal temperature and water po-

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tential of the whole population in a single equation by incorporating a suit-able distribution (usually a normal distribution) of base water potentialswithin the population (Gummerson, 1986; Dahal and Bradford, 1994;Dahal, Bradford, and Haigh, 1993; Bradford, 1995). A form analogous toEquation 2.8 gives:

Probit (G) = {[ – HT / (T – Tb) t(G)] – b(50)} / b (2.11)

The best fit to the model can be obtained by repeated probit regressionsvarying the values of HT. Following this approach used by Bradford (e.g.,Bradford, 1995), the time courses of germination at suboptimal tempera-tures and water potentials can be mapped on to a common scale by multi-plying time to germination [t(G)] by the factor {1 – [ / b (G)]} (T – Tb). Ina range of tomato seed lots the hydrothermal time model accounted for 73 to93 percent of the variation in radicle emergence timing across a range oftemperatures and water potentials (Cheng and Bradford, 1999).

To be useful for field predictions the hydrothermal time model must alsobe able to describe the reduction in germination rate and nongerminationthat occurs at supraoptimal temperatures. So far it has been assumed that thefive parameters [ HT, Tb, b(50), b, and the fraction of viable seeds (Gm)],which can describe behavior of the whole seed population, are constant.However, Bradford (1995) suggested that progressive loss of dormancy, as-sociated with increased percentage germination and germination rate, in aseed population may be related to a progressive decrease in b(50). Chris-tensen, Meyer, and Allen (1996) demonstated that changes in the germina-tion time courses of Bromus tectorum seeds during after-ripening could befully accounted for by changes in b(50). Therefore as the time in storage(after-ripening) increased, the distribution of b remained the same in theseed population, but their water potential thresholds were reduced belowthat of the ambient water potential to allow germination. As after-ripeningcontinued, their b was further reduced below that of ambient levels andgermination rate increased. During thermoinhibition of lettuce (Lactucasativa) the reverse occurred and the water potential thresholds increased astemperature approached the upper limit for germination (Bradford andSomasco, 1994). In this case, thresholds shifted above ambient water poten-tial preventing germination. Subsequently, other studies have shown that astemperatures become supraoptimal and approach Tc, the b distributionshifts progressively toward and above 0 MPa to reduce germination rate andeventually prevent germination (Figure 2.4a; Kebreab and Murdoch, 1999;Meyer, Debaene-Gill, and Allen, 2000; Bradford, 2002). In this way, germi-nation time courses can be accounted for over the whole temperature range.

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The distribution of b accounting for variation in germination times withinthe population remains the same, but above the optimum temperature (Topt),

b(G) increases linearly with T (Figure 2.4a). This in turn accounts for theparallel decreases in germination rate above the optimum and the distribu-tion of ceiling temperatures discussed in relation to Equation 2.3 (Figure2.2a). Therefore, Equation 2.10 was modified by Bradford (2002) to ac-count for the germination response to supraoptimal temperatures:

HT b opt T opt opt bG k T T T T t G (2.12)

Ger

min

atio

n ra

te[1

/t(50

)]�

b(M

pa)

Temperature( C)�

a) b)Topt T Td opt

Tb TbTc Tc

FIGURE 2.4. Schematic representation of the relationship between b(50) andtemperature and their effect on the germination rate of percentile G50. (a) b(G)initially remains constant as temperature increases above Tb, however b(G)increases at temperatures above the optimum (Topt), as in Equation 2.12, toexplain the reduction in germination rate. At Tc germination is prevented as

b(G) increases above 0 MPa. (b) b(G) increases from the deviation tempera-ture (Td) in advance of (Topt) as in the temperature modification described forEquations 2.15 and 2.16. In both situations, a and b, the distribution of b(G)around b(50) remains the same and this results in the same distribution for Tc(Equation 2.6).

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where kT is a constant (the slope of the b(G) versus T line when T > Topt)and b(G)opt is the threshold distribution at Topt. This equation adjusts

b(G) opt to higher values as T increases above Topt. Since the standard de-viation of the b(G) distribution is not affected, the b values of all of theseeds are adjusted upward by the same amount for each increment in Tabove Topt. Equation 2.12 also stops the accumulation of thermal time at thevalue equivalent to that accumulated at Topt. Thus, temperatures above Toptdo not contribute additional thermal time in the supraoptimal range. In-stead, effects on germination are accounted for by the change in b(G).

By combining Equation 2.10 for suboptimal temperatures and Equation2.12 for the supraoptimal temperatures, seed germination time courses canbe described in hydrothermal time for all temperatures. Battaglia (1997)uses an alternative, and more flexible, linear predictor technique based on asimilar conceptual framework that could incorporate responses to sub- andsupraoptimal temperatures as well as other factors. However, the linear pre-dictor assumes that variation in germination rate results from the distribu-tion of the factor threshold (base or ceiling). It therefore accommodatesEquations 2.3 (supraoptimal temperature) and 2.7 (water potential), but notEquation 2.3 (suboptimal temperature) where base temperature is assumedto be a constant.

Seed Advancement Below Base Water Potential

The models described previously have been used widely and success-fully to describe data collected under laboratory conditions of constant tem-perature and water potential. Within the hydrotime and hydrothermal timemodels germination is arrested by water potentials that fall below b. How-ever, it is known from priming studies that metabolism continues below wa-ter potentials which prevent the completion of germination and radiclegrowth. After such a period of priming, germination is more rapid. Themodels described earlier do not take account of this advancement. There-fore under seedbed conditions where water potential varies above and be-low b, germination time will be overestimated because no hydrotime orhydrothermal time is accumulated below b. In addition to priming, suc-cessive wetting and drying of the seeds under field conditions can also sig-nificantly advance germination (e.g., Wilson, 1973; Koller and Hadas,1982; Hegarty, 1978; Allen, White, and Markhart, 1993; Adams, 1999;González-Zertuche et al., 2001). Two approaches are currently being devel-oped to account for seed advancement below b.

In the first approach, the concept of hydropriming time was developedbased upon the principles developed previously in this chapter (Tarquis and

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Bradford, 1992). During priming, seeds accumulate hydropriming time inproportion to the difference between the water potential of the priming so-lution and the minimum required for metabolic advancement to occur dur-ing priming ( min). In this treatment the solution is always below b. Forlettuce, Tarquis and Bradford (1992) estimated that the median b was –1.0Mpa and min was –2.4 MPa. A very similar min has also been estimatedfor a range of tomato seed lots (Cheng and Bradford, 1999), but values be-tween –5.0 and –8.0 Mpa were recorded for Elymus elymoides (Meyer,Debaene-Gill, and Allen, 2000). In the model, germination rates after prim-ing are assumed to increase linearly with any increase in accumulatedhydropriming time thus:

GR50 = GRi + k ( – min) tp (2.13)

where GR50 is the median germination rate (1/ t50) of primed seeds, GRi isthe initial median germination rate before priming, and tp is the duration ofthe priming treatment at water potential , and k is a linear proportionalityconstant. (Tarquis and Bradford, 1992; Bradford and Haigh, 1994; Brad-ford, 1995). However, in the seedbed, such advancement at water potentialsbelow b will not occur at constant temperatures as they do in priming andso this concept was extended to include accumulated thermal time as hydro-thermal priming time (Bradford and Haigh, 1994; Bradford, 1995). In thiscase germination rate after priming can be expressed as

GR50 = GRi + k1 (T – Tmin) ( – min) tp (2.14)

where Tmin is the minimum temperature at which a priming effect will occurand k1 is a proportionality constant. Hydrothermal priming time [(T – Tmin)( – min) tp] will accumulate in proportion to the extent by which ex-ceeds min and T exceeds Tmin. Under laboratory conditions this approachwas able to describe the effect of priming on germination rates (Cheng andBradford, 1999; Meyer, Debaene-Gill, and Allen, 2000). In these primingmodels, a single value for min has been used, but it may vary within theseed population with the effect of increasing variation in germination times.Bradford (1995) speculates that in a situation where water potential variesacross the range of base and minimum water potentials hydrothermal andhydrothermal priming time could be used additively in some form to predictgermination times.

A potential limitation to this approach was pointed out by Rowse,McKee, and Higgs (1999) in that it necessarily predicts that the increase ingermination rate is proportional to tp, whereas, in practice it tends to reach a

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maximum and then does not increase further with increased priming time.Rowse, McKee, and Higgs (1999) have developed an alternative model thatcan describe the effects of fixed and variable water potentials, both aboveand below b. The model is loosely based on the idea that for a seed to initi-ate radicle growth its cells have to generate sufficient turgor pressure to ex-ceed the yield threshold (Y) for growth. The model arbitrarily assumes thatY remains constant and turgor is determined in the cells by changes in os-motic potential and by changes in external water potential. Within themodel, values of osmotic potential are empirical and they are thereforetermed virtual (VOP) and assigned the symbol . The model determinesseed advancement to germination by integrating changes in that areproportional to the history of water potential experienced by the seed rela-tive to minimum and base water potentials. The minimum ( min) definesthe water potential below which there is no metabolic advancement (prim-ing) and the base ( b) defines the water potential above which radiclegrowth can occur. According to the model, germination time for a givenconstant suboptimal temperature [t(G, T)] and water potential can be deter-mined by

t G Tk T

G Y

G

b,

/ln

min

1

10

(2.15)

where k0 (T) is the rate constant when = 0. To fit the model, b, as in thehydrotime model, is assumed to have a normal distribution. The VOP modelcan be used in finite difference simulation to calculate changes in andpredict germination when varies thus:

d G dt k T G Y Gv b v/ / min0 1 (2.16)

The VOP model has now been extended to include temperature (Rowse,personal communication). At temperatures where the germination rate isproportional to (T – Tb), this is done by assuming that the rate constant isproportional to hydrothermal time [e.e., the terms k0(T)(1 – / min) inEquations 2.15 and 2.16 are replaced by k(T/Tb – 1)(1 – / min]. Experi-ments on carrot and onion seed (Rowse, unpublished) have shown thatabove a critical temperature (Td) the germination rate ceases to be linearlyrelated to temperature (Figure 2.4b). This situation can be well accommo-dated by assuming that b changes so that for any seed fraction the effectivebase water potential is given by b + m(T – Td), where b is the uncor-rected base water potential and m is a coefficient. Thus b increases athigher temperatures as described for hydrothermal time (Equation 2.12);

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however, note that Td is well below the temperature when the germinationrate is a maximum (Figure 2.4b). For carrot and onion the fitted values areapproximately 18 and 16oC, respectively, whereas maximum germinationrate occurred close to 25oC. Thus increase in rate due to T – Tb between Tdand the optimum is offset by an increase in b reducing – b. In this waya curved response results in contrast to that of hydrothermal time. Using thisapproach the model can be used to predict germination in conditions thatvary above and below b at the full range of temperatures. This same ap-proach can also be used effectively to take account of supraoptimal temper-atures in the hydrothermal time model (Rowse, pers comm).

The VOP model utilizes the concepts of base and minimum water poten-tials developed in hydrothermal time models; however, it has a differentialformulation and does not assume that a seed must be in either a germinatingor a priming state (water potential is treated as a continuous variable abovethe minimum water potential). Such a model is potentially very useful forpredictions under variable seedbed conditions but has yet to be tested on arange of seed lots and conditions. In contrast, hydrothermal and hydrother-mal priming time models have been tested and found to be descriptive on awider range of seed lots but do not lend themselves so readily to predictionunder variable conditions in their present form. The application of thesemodels for field prediction is considered in the next section.

Further Development of Threshold Models

The threshold models described previously provide a robust frameworkin which to describe seed responses to the environment. Even though thesemodels are fitted empirically, the thresholds determined appear to have aphysiological basis. However, it is important to appreciate that at presentthese models do not account for all seed behavior and further developmentis necessary to incorporate sufficient flexibility to cover the extent of bio-logical variability scientists have come to expect. Much of this variabilitymay result from interactions between and T resulting in concurrentchanges in thermal and hydrotime parameters. In addition, physiologicaladaptation occurring near both b and Tb resulting in greater than expectedgermination rates and percentage germination may result from overlap ofwhat we now consider to be separate priming and germination processes.As a greater range of species are investigated the discrete packaging ofthese model components, although convenient, may cease to be appropri-ate.

For example, the comprehensive data set and analysis conducted byLabouriau and Osborn (1984) on tomato seeds shows linear relationships

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between germination rate and temperature in both sub- and supraoptimalranges. However, the optimum occurred over a range of temperatures be-tween 25.9 and 29.5 rather than a sharply defined optimum at the conver-gence of the two linear relationships as used by Garcia-Huidobro, Mon-teith, and Squire (1982a) and Covell and colleagues (1986) for otherspecies. This plateau can be accommodated in a further development of thethermal time model based on Gaussian curves which describes the germina-tion response across both sub- and supraoptimal temperature ranges (Orozco-Segovia et al., 1996). However, the optimum temperature can also differwith water potential (Kebreab and Murdoch, 2000). Responses to both tem-perature and water potential can be accommodated within the hydrothermaltime model by an increase in b with temperature in advance of Topt. In thiscase, the increased rate of hydrothermal time accumulation resulting fromhigher temperature (i.e., increase in T – Tb) as the optimum is approachedwould be offset by a concurrent increase in b (reducing – b). Data re-ported for fully after-ripened seeds of Elymus elymoides (Meyer, Debaene-Gill, and Allen, 2000) and observations in onion and carrot (Rowse, per-sonal communication) can be explained in this way. For, example, in fullyafter-ripened Elymus elymoides seeds, b increased linearly with tempera-ture (10 to 30 C), resulting in little difference in germination rates over thisrange of temperature (Meyer, Debaene-Gill, and Allen, 2000). Kebreab andMurdoch (1999) have also shown that in Orobanche aegyptiaca seeds theunderlying assumption of independance of and T effects within the cur-rent hydrothermal time model is not valid, and they give examples of workwith other species where this is also the case. They found that b variedwith T, both above and below the optimum, and Tb varied with and devel-oped a new and more general thermal time model that allows for the interac-tion of temperature and base water potential (Kebreab and Murdoch, 1999,2000). Alternatively, the approach described by Battaglia (1997) can incor-porate complex factor interactions and test them for significance in affect-ing the germination response. However, it has yet to be seen how well thehydrothermal time model, freed from the constraints of fixed thresholds,can account for the full range of seed responses to environment that are re-ported in the literature. Other current concerns, such as nonlinear relation-ships close to Tb outlined as follows, may also be reconciled in this way.

It is generally accepted that, when calculated by linear rate temperaturerelationships, there is a single base temperature below which germinationof the whole population will not occur. Extrapolation of a linear relation-ship covering suboptimal temperatures of 10°C and above indicates a singlebase temperature in tomato (e.g., Dahal, Bradford, and Jones, 1990, and ref-erences within). Yet Labouriau and Osborn (1984), for example, show thatpercentage germination declines progressively over the range 10 to 6°C, in-

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dicating a range of thresholds within the seed population as seen in manystudies. If this apparent dilemma is considered in terms of residual dor-mancy, expressed close to the base temperature, it could be accommodatedin a modeling approach developed for species with seasonal changes in therange of temperatures which permit germination (Washitani and Takenaka,1984; Washitani, 1987; Kruk and Benech-Arnold, 1998, 2000; Chapter 8).In this approach, Tb is used to calculate rates in thermal time as describedpreviously, but seeds also have a lower temperature (Tl) below which germi-nation is prevented by dormancy. Tl is assumed to have a normal distribu-tion within the population. Thus percentage germination declines over arange of temperatures as Tb is approached. In weed species, Tl and an equiv-alent higher limit temperature (Th) can change during the season as temper-ature changes to account for seasonal dormancy patterns. In genetically uni-form crop seeds, produced without residual dormancy (i.e., Tl = Tb), a singlebase temperature may well be sufficiently accurate for predictions of germi-nation, whereas in a seed lot from mixed populations or uncultivated spe-cies this is less likely to be the case. In fact, a normal distribution of mini-mum as well as maximum temperature thresholds is seen widely in theliterature (e.g., Grundy et al., 2000). In addition, consistent deviations fromthe linear relationship between rate and temperature at suboptimal tempera-tures can occur in some crop species close to Tb. This behavior can severelyaffect the prediction of germination time at constant temperature close tothe base (Marshall and Squire, 1996; Phelps and Finch-Savage, 1997).

Kebreab and Murdoch (2000) and Grundy and colleagues (2000) havedeveloped separate modeling approaches to incorporate independent seedto seed variation in both minimum and maximum temperature thresholdswithin the general conceptual framework discussed here. These approachessuggest that thresholds and rates can behave independently, so they involvethe separate determination of germination rates and final percentage germi-nation. One advantage is that rate relationships within the threshold model-ing approach are not constrained to be linear if this limits the precision re-quired for field prediction (Grundy et al., 2000). Indeed, linear relationshipbetween temperature and development have often been shown to occurwithin a limited temperature range only, and in many other biological sys-tems rates are more often described by nonlinear relationships (Sharpe andDeMichelle, 1977; Schoolfield, Sharpe, and Magnuson, 1981). The mathe-matical approach adopted by Sharpe and DeMichelle (1977) closely fits ob-served data and accommodates the linearity in response over a limited tem-perature range that has been adopted in thermal time models. There is,however, a practical disadvantage. The determination of bases explicitly (fi-nal number that germinate) is time consuming, as germination inevitablytakes a considerable time under conditions close to temperature or water

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potential thresholds and there is considerable risk of achieving a poor esti-mate by early termination of the experiment or the intervention of contami-nation. If curved responses between germination rate and temperature andvariation in Tb can be accommodated within the hydrothermal time modelby changes in b these concerns could be eliminated. Further work is re-quired to determine whether the hydrothermal time model can be suffi-ciently flexible to accommodate the full range of seed behavior.

It is common knowledge that time spent below b (e.g., Kahn, 1992) andTb (Coolbear, Francis, and Grierson, 1984) can increase subsequent germi-nation rates when seeds are placed above these thresholds. Progress aboveand below these thresholds are not directly additive and there is not a clearpredictive relationship between hydrothermal and hydrothermal primingtime models (Cheng and Bradford, 1999), suggesting that they are separateprocesses. There is no obvious reason to consider these processes as mutu-ally exclusive. Indeed, seed characteristics can change in constant condi-tions above b; for example, there is evidence that extended incubation ofseeds between –0.5 MPa and b results in a shift to lower values of b (Niand Bradford, 1992; Dahal and Bradford, 1994). This adaptation at constantlow temperature may account for some observations of nonlinear behaviorclose to the threshold in laboratory experiments. In variable field environ-ments such prolonged exposure to a particular set of conditions is unlikely,so field prediction may not be affected by this behavior.

Garcia-Huidobro, Monteith, and Squire (1982b) point out that for thresh-old models developed from constant environments, several conditions needto be satisfied before they can be used in variable conditions. These include(1) the instantaneous rate of development should depend only on the currentconditions and not their history of exposure and (2) values such as thermaltime and base temperature should remain unaffected. In nondormant lentilseeds, at least at suboptimal temperatures, there was no effect of thermalhistory on germination rate and thermal time could be used to predict timerequired for germination at alternating temperatures (Ellis and Barrett,1994). However, when seeds have residual dormancy then there can be sys-tematic deviation from predictions resulting from their history of exposure.For example, exposure to alternating temperatures can have a positive effecton rate of germination (Garcia-Huidobro, Monteith, and Squire,1982b) andpercentage germination (Murdoch, Roberts, and Goedert, 1989), whereasexposure to high temperatures can have a negative impact on these samemeasures (Garcia-Huidobro, Monteith, and Squire,1982b). Although it isconvenient to consider crop species as nondormant, deviation of their be-havior in some cases from that readily described by the commonly used hy-drothermal time model may result from limited residual dormancy. This isin keeping with the view that differences in crop seed performance (vigor)

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may be an extension of dormant behavior toward the end of a continuousscale (Hillhorst and Toorop, 1997).

The question to ask is, Do these current limitations of the hydrothermaltime model have practical significance? For many purposes, such as com-parison of genotypes or treatments (e.g., Covell et al., 1986; Dahal, Brad-ford, and Jones, 1990) this approach is very effective. For field predictionpurposes, errors at low temperature are likely to have an impact in early sea-son crops grown at suboptimal temperatures, but inaccuracy is likely to belimited, especially as ambient temperatures rise in the spring followingsowing. However, errors in prediction close to the optimum temperature,when progress is rapid, can have a major impact on the prediction of germi-nation and emergence in clock time.

OTHER GERMINATION MODELS

In the present work, population-based threshold models have been usedto describe responses to the environmental because it is likely that they havephysiological significance and provide a framework for developing a ge-neric understanding of seed and seedling responses. However, for seedlingemergence prediction in the field other modeling approaches can have sig-nificant merit, but here there is not space to do these models justice. A largenumber of models have been developed to describe germination and emer-gence responses (e.g., Wanjura, Buxton, and Stapleton, 1970; Blacklow,1972; Scott, Jones, and Williams, 1984; Thornley, 1986; Forcella, 1993;King and Oliver, 1994; Hageseth and Young, 1994; Pemberton and Clif-ford, 1994; Gan, Stobbe, and Njue,1996). There are also more mechanisticapproaches, for example, that of Bruckler (Bruckler, 1983a,b; Bouaziz andBruckler, 1989a,b) and Dürr and colleagues (2001). A number of thesemodeling approaches have been reviewed by Forcella and colleagues (2000).

POSTGERMINATION SEEDLING GROWTH

Some monocot preemergent seedlings are resistant to desiccation due totheir seminal root system that can readily replace damaged roots. However,in the majority of crop species, once the seed has initiated growth the grow-ing seedlings progressively become desiccation sensitive and therefore arecommitted to continued growth (Bewley and Black, 1978). It is an obviouscomment, but postgermination growth occurs in two directions; the patternin which it does this is essential for survival and also for prediction of emer-gence time. Rapid downward growth is necessary to maintain contact with

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moisture in the seedbed as it dries from the surface. Growth upward, toreach light and establish an autotrophic seedling, usually occurs in a deteri-orating seedbed (increasing impedance to growth) and must be completedbefore seed reserves are exhausted.

Close to the soil surface, germination tends to occur most often afterrainfall. For example, not only is germination metabolism (as shown previ-ously for priming) less sensitive to water potential than the initiation ofgrowth, so is postgermination extension growth (Ross and Hegarty, 1979).Initiation of growth is therefore a moisture-sensitive, rate-limiting step thatdetermines b and ensures that in many species germination under variablesoil conditions occurs only when sufficient moisture is likely to be availablefor subsequent seedling growth (Hegarty, 1977; Ross and Hegarty, 1979;Finch-Savage and Phelps, 1993, Finch-Savage, Steckel, and Phelps, 1998).Below b, seed priming or advancement in the soil (Wilson, 1973; Allen,White, and Markhart, 1993; Rowse, McKee, and Higgs, 1999) means thatgermination can be rapid when water becomes available. In the absence ofadditional water, there is only a brief opportunity for the completion of ger-mination and seedling growth before the surface soil layers dry again. Fol-lowing germination, initial growth is downward in both epigeal and hypo-geal seedlings, to maintain contact with soil moisture as it dries from thesurface layers. As this drying occurs the hydraulic conductivity of soil in thesurface layer quickly falls to a very low value, and this will tend to reducethe rate of water loss from deeper layers (e.g., Lascano and van Babel,1986). The seedling root will therefore grow into increasingly wet soil andthe seedling may become less dependent on moisture content of the surfacelayers (Bierhuizen and Feddes, 1973). This pattern may occur becausehypocotyl extension is more sensitive than radicle extension to low matricpotential which initially favors the growth of roots (Dracup, Davies, andTapscott, 1993). The initial period of downward seedling growth followinggermination is therefore critical to successful seedling establishment. Up-ward growth often occurs in a deteriorating seedbed that has increasing soilstrength. Even if water potential is not directly limiting, because the grow-ing root maintains contact with adequate moisture, there can be a large indi-rect effect because soil strength above the seed will increase as water con-tent decreases. Thus in practice, during the postgermination phase of cropemergence, mechanical impedance may have greater importance than waterstress in delaying and reducing the number of seedlings emerging (Whalleyet al., 1999). In addition, soil can become much stronger following rainfalleven without subsequent drying (Hegarty and Royle, 1976, 1978).

A large number of studies have been made of the response of preemer-gence seedling growth to temperature (e.g., Wanjura, Buxton, and Staple-ton, 1970; Blacklow, 1972; Hsu, Nelson, and Chow, 1984; Wheeler and

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Ellis, 1991; Weaich, Bristow, and Cass, 1996; Vleeshouwers, 1997; Romanet al., 1999; Shrestha et al., 1999). Different methods have been used to de-scribe growth data from different species, but in many cases a thermal timeapproach similar to that described for germination has been adopted whichassumes growth rate is linearly related to temperature. However, the utilityof thermal time and other techniques that account only for temperature havelimited potential in practice for accurate crop emergence predictions be-cause soil moisture and strength vary greatly in the surface layers of the soil.Fewer studies have been made on the interaction between temperature andwater potential (e.g., Fyfield and Gregory, 1989; Choinski and Tuohy,1991; Dracup, Davies, and Tapscott, 1993) or soil mechanical resistance topreemergence seedling growth (e.g., Mullins et al., 1996; Vleeshouwers,1997; Vleeshouwers and Kropff, 2000). A review of this literature is notjustified in the present work which has its emphasis on germination.

Recently a model was developed that incorporates the effects of the threeubiquitous seedbed factors, temperature, water potential, and soil imped-ance, on preemergence shoot growth (Whalley et al., 1999). The model (de-scribed in the Appendix) assumes a linear dependence on temperature andwater potential and scales the basic thermal time model so that shoot elon-gation rate decreases proportionally as impedance increases and water po-tential decreases toward threshold values that will just stop elongation. Themodel has a single threshold for each factor and so describes mean shootgrowth only (Whalley et al., 1999). Variation in elongation rates has beenintroduced into predictive models by assuming a normal distribution ofrates within the population (Finch-Savage and Phelps, 1993; Vleeshouwersand Kropff, 2000). Finch-Savage and colleagues (2001) have developed amodel that describes a distribution of temperature and water potentialthresholds for preemergence growth to take account of variation within thepopulation. For prediction, nonemergence can be modeled by incorporatingseed weight and the exhaustion of reserves with time, in particular as emer-gence time is extended by soil resistance to growth (Whalley et al., 1999;Vleeshouwers and Kropff, 2000). Another factor to be considered is the ef-fect of seedbed structure (Bouaziz and Bruckler, 1989b; Mullins et al.,1996; Dürr et al., 2001).

THRESHOLD MODELS: PREDICTION OF GERMINATIONAND EMERGENCE PATTERNS IN THE FIELD

Forcella and colleagues (2000) have reviewed the use of thermal time inseedling emergence prediction and shown that in many circumstances it canbe very effective. Bierhuizen and Feddes (1973) show that the heat sum ap-

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proach can accurately predict time to 50 percent emergence of vegetablecrops provided soil moisture content is taken into account. They suggestthat in dry periods a quantity of 5 to 10 mm of irrigation should be givenregularly, which will shorten the period to emergence and avoid the impactof crust formation. Indeed, heat sums can be used to time irrigation to bettereffect to achieve the same purpose (Finch-Savage, 1990a,b). However, inthe absence of irrigation, soil moisture varies greatly in the surface layers ofthe soil and so thermal time has limited ability to predict emergence. In bothuntreated seed and seed after advancing treatments (e.g., priming) the tim-ing of water availability in the surface layers of the seedbed and its effect ongermination can be the main factor determining time to seedling emergencein crops with nondormant seeds (HåKansson and von Polgár, 1984; Finch-Savage, 1984a,b, 1987, 1990a). In the case of onions, seedling emergencewas reduced or delayed by inadequate soil moisture on more than half of 45sowings made over three years (Roberts, 1984). Following germination,particularly during upward growth of the shoot, it has been argued that soilimpedance is likely to become the principal factor. Seedbed conditions aredescribed in detail in Chapter 1 and will only be discussed here as they im-mediately relate to germination and preemergence seedling growth.

The relatively recent and continuing development of threshold models,describing both temperature and water potential effects on germination andpreemergence growth, has so far resulted in few attempts to apply thesetechniques under variable field conditions. Hydrothermal time approacheshave been used with some success to describe the timing of major flushes incrop seedling emergence (Finch-Savage and Phelps, 1993; Finch-Savage,Steckel, and Phelps, 1998; Finch-Savage et al., 2000) and that of weeds(Battaglia, 1997; Bauer, Meyer, and Allen, 1998; Roman, Murphy, andSwanton, 2000). However, the first section of this review illustrates the im-portance of being able to predict the uniformity and numbers of seedlingsemerging as well. The remainder of this section will be used to illustrateseed responses in a field context to show how variation in seedling emer-gence is generated and how the models described can be applied to predictcrop seedling emergence.

A Modeling Framework

Threshold models for germination and preemergence seedling growthcan be used to simulate seed germination and seedling emergence by divid-ing time into manageable steps and applying the models to each step. Themodel time (e.g., thermal, hydrothermal time, VOP, etc.) is accumulatedfrom each step to indicate progress toward seedling emergence in clock

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time within the seed population. A schematic illustration of the process ofsimulation is provided in Figure 2.5, showing separate submodels for seed-bed conditions, germination, and preemergence seedling growth. The ger-mination and preemergence growth submodels are driven by appropriateoutputs from the seedbed model calculated from details of the soil and me-

Plant Soil

Sowseed

Seedalive?

Deadseed

Increment seeddevelopment

Able togerminate?

Increment root growthIncrement shoot growth

Seedlingstate?

Deadseedling

Emergedseedling

At seed depthTemperatureWater potential

Profile ofTemperatureWater potentialSoil strength

FIGURE 2.5. Flow diagram to illustrate the components of the simulation modelused in Figure 2.6 (Source: Reproduced from W. E. Finch-Savage, 2003, Vege-table seed vigour: Looking to the future, The Vegetable Farmer, January, pp. 28-29, with permission from ACT Publishing.)

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teorological data. The major drivers for germination are temperature andwater potential, and as each seed in the population germinates, soil strengthand its resistance to growth also become important. However, in the surfacelayers where the seeds are sown there can be steep profiles of all three ofthese variables, with temperature declining and moisture content increasingbelow the surface. In addition, fluctuations are dampened at increasingseedbed depths. To further complicate the situation, seeds are rarely sown atuniform depth, and irregularity in soil aggregate size will vary seed contactwith the soil, so seeds within the population will experience different condi-tions. For simulation purposes this situation is accounted for by assigningdepth and germination characteristics (e.g., base temperature and water po-tentials), drawn at random from the appropriate frequency distribution, toeach seed. The models are then run for each seed using inputs appropriatefor that depth. Increasing the number of individual seeds will generate amore reproducible prediction of seedling emergence times in the popula-tion. Following germination, as the seedlings grow, it is necessary to decidewhich temperature and water potential down the profile is most appropriateto use as inputs into the models. There is little guidance in the literature, sothese decisions are largely pragmatic.

Although the measurement and simulation of seedbed conditions are de-scribed elsewhere (Chapter 1 and reviewed by Guérif et al., 2001), it is nec-essary to mention them here, in general terms, as the accuracy of these mea-surements determines the success of the simulation. These measurementsarguably present the major limitation to predicting seedling emergence. Forexample, direct measurement of water potential at seed-scale resolution isnot possible and so must itself be modeled from soil water content using awater release curve. When soils dry, water potential varies with water con-tent according to a power law so that a small error in the measured watercontent can lead to a large error in the predicted water potential. Relevantsoil water content measurement at seed-scale resolution is also difficult,even with more sophisticated techniques such as time-domain reflectome-try (TDR). This equipment is sensitive to soil bulk density, so even in care-ful field experiments it is difficult to know which calibration to use (Whal-ley, 1993). Therefore, reliable water potential estimates are difficult toachieve and small differences can have a large impact upon the prediction ofemergence time in a drying seedbed. Even for temperature, for which mea-surements at seed-scale resolution are possible, usually only a single soil orair temperature is available, so the temperature profile in the seedbed mustbe modeled. Prediction of seedling emergence will be only as good as thesemodels of seedbed conditions.

Modeling the seedbed environment in any detail is very difficult, never-theless models of differing complexity have been developed to provide rele-

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vant variables for seedling emergence models (e.g., Walker and Barnes,1981; Forcella, 1993, 1998; Mullins et al., 1996; Brisson et al., 1998; Roman,Murphy, and Swanton, 2000; Dürr et al., 2001). These seedbed models canbe coupled with threshold models that indicate how seeds and seedlings canrespond to environment, to predict seedling emergence (Figure 2.5).

Germination

The following section is concerned with the prediction of crop seed ger-mination and therefore the impact of environmental conditions on dormancystatus will not be considered here (see Chapter 8). However, the applicationof hydrothermal time to the description and prediction of dormancy statusin weeds has recently been reviewed elsewhere (Bradford, 2002). Undervariable seedbed conditions, thresholds can be interpreted as switches fornondormant seeds: germination either proceeds or stops according to thevalue of ambient conditions relative to the threshold (Figure 2.6). Thus, rateof progress toward germination decreases and less physiological time (hy-drothermal or hydrothermal priming time or VOP) is accumulated for agiven clock time as min and/or Tmin and Tc are approached. Below b or Tb,and above Tc radicle emergence will not occur.

It is likely that these thresholds have physiological significance relatingto the initiation of radicle extension (Welbaum et al., 1998; Meyer, Debaene-Gill, and Allen, 2000; Bradford, 2002) and operate as rate-limiting steps inthe progress of seedling emergence from the soil. For example, the interac-tion of b and the changing amount of soil moisture can largely determinethe timing of onion seedling emergence in the field (Finch-Savage andPhelps, 1993). Such a mechanism should avoid germination into seedbedconditions likely to be hostile to subsequent seedling growth.

The threshold models described can be applied in dynamic forms in fi-nite difference simulation to predict progress toward germination above

min. The VOP model (Equation 2.16), with the modifications discussed forthe effects of temperature, can now be applied directly to describe field ger-mination (Rowse, personal communication; Figure 2.6), whereas a differ-ential form of the hydrothermal time model covering both sub- and supra-optimal temperatures (Equations 2.10 and 2.12, respectively) must alsosomehow be coupled to hydrothermal priming time (Equation 2.14) to takeaccount of progress that occurs below b. One approach to the latter was at-tempted by Finch-Savage and colleagues (2000) who interpreted the equa-tion suggested by Bradford (1995), and later modified (Bradford, 2002), tosum progress in both hydrothermal and hydrothermal priming time. How-ever, the more flexible modeling approach used by Battaglia (1997) capable

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FIGURE 2.6. Simulation of the effects of soil temperature (a) and soil waterpotential (b, d) on cumulative germination and seedling emergence of onion(c, e) at two sowing depths (Rowse, Whalley, and Finch-Savage, unpublished).Seeds were sown at 9 ± 1 mm (b, c) or 19 ± 1mm (d, e).The sowing was made onJune 13, 1996, at Wellesbourne, United Kingdom, in a sandy loam soil. Temper-atures were measured at sowing depth and the water potential profiles were sim-ulated from standard meteorological data and soil characteristics. There wassome difference in temperature at the two depths, but for brevity only 9 mm isshown. Germination (dashed line) and emergence (solid line) times were pre-dicted using models published by Rowse, McKee, and Higgs (1999) (Equation2.16 with temperature modifications described previously) and Whalley and col-leagues (1999, Appendix) respectively. Closed circles are observed seedlingemergence. Temperature and water potential thresholds used in the simulationare shown. The predictions were made on 100 individual seeds each assigned asowing depth, b and Tb at random from distributions determined for the seedpopulation. This simulation is being constructed with the future aim of demon-strating the consequences of environmental conditions and grower interventionson germination and seedling emergence.

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of including a range of factors and their interactions may prove to be an ef-fective way of applying threshold models to variable field conditions. Usingthis approach, reasonable prediction of Eucalyptus delegatensis seed ger-mination in the field was possible (Battaglia, 1997). It is relevant to pointout that in contrast to crop species, germination of the Eucalyptus dele-gatensis seed population was spread over months in these experiments. Un-der these circumstances the accuracy of soil water prediction discussed ear-lier has less importance.

The median b for lettuce has been estimated by Tarquis and Bradford(1992) to be –1.0 MPa and min as –2.4 MPa. Similar values have now beendetermined for a number of crop species. In practice, water potential in thesurface layers of the seedbed can change quickly (e.g., Figure 2.6) so thatlittle clock time is spent between these water potential thresholds. Thus rea-sonable predictions of seedling emergence time, under many seedbed con-ditions, may be possible without including advancement below b. Indeed,the timing of flushes of onion seedling emergence can be described reason-ably well using a modified hydrothermal time model which assumed thatgermination progressed in thermal time (unaffected by ) above b andprogress ceases below it (Finch-Savage and Phelps, 1993). A similar ap-proach has been used to predict the timing of weed seedling flushes withsome success (Forcella, 1998; Forcella et al., 2000). This approach worksbecause soil water potential rises instantly upon rain and then, at shallowsowing depth, falls rapidly to levels below which metabolic advancementoccurs in the seed. This clear pattern of soil water potential can mask the in-accuracies of the simple model used, but the success of the model illustratesthe importance of the rate-limiting moisture-sensitive step ( b). Neverthe-less, in a further set of field experiments, this model described carrot germi-nation more accurately than the hydrothermal model which overestimatedtime to germination (Finch-Savage, Steckel, and Phelps, 1998) when soilmoisture was limiting. Similarly, Roman, Murphy, and Swanton (2000) ac-curately predicted seedling emergence of Chenopodium album in springunder no-till conditions using hydrothermal time to account for germina-tion in the model. However, in seedbeds that were cultivated and thereforemore subject to drying, the model overestimated time to emergence. Onereason for overestimation could have been that progress below b was notconsidered. An attempt to account for hydrothermal priming time in the car-rot experiments improved prediction of germination times in dry condi-tions, but time to germination was still overestimated (Finch-Savage et al.,2000).

It has been argued previously that the most likely cause of overestima-tion of the time to germination results from inaccurate estimates of soil wa-ter potential at sowing depth. However, there are a number of other reasons

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why this may have occurred. There may have been a catastrophic effect; forexample, rapid drying imposed shortly before radicle emergence can altergermination rates upon return to water (Debaene-Gill, Allen, and White,1994). Alternatively, overestimation of germination time suggests thatseeds progressed toward germination faster in variable field conditions thanwould be expected from laboratory experiments under constant conditions.As discussed earlier, seeds have been shown to adapt physiologically toprolonged exposure to low water potentials by lowering b (Ni and Brad-ford, 1992). There is also an implicit assumption in the models that seedswet up and dry as rapidly and to the same extent as the surrounding soil andthat the impact of this on the rate of progress toward germination, includingradicle emergence, is equal throughout the germination process. However,seeds may resist water loss so that they do not dry as quickly as the sur-rounding soil or they may acquire water that condenses on them in a cyclingtemperature environment. Moisture in the vapor phase can also be an im-portant component of seed germination in the field (Bruckler, 1983a,b;Wuest, Albrecht, and Skirvin, 1999). The seed response may not remain thesame throughout all stages of germination. It is also possible that once theseeds have become sufficiently hydrated they subsequently germinate fasterat suboptimal water potentials than if they were imbibed and remained atthat water potential, as in constant laboratory conditions. These possibili-ties could be resolved by experimentation using controlled changes in ex-ternal water potential and temperature; however, such experimental data islacking in the literature.

In horticulture, it is reasonable to assume that seeds are sown into ade-quate moisture (Finch-Savage and Phelps, 1993) for initial imbibition.However, this will not be the case in all situations or with other crops. A fur-ther potential error is that the models assume instantaneous reaction tochanges in seedbed conditions; as discussed previously, this may not be truein the case of water potential changes. For prediction, imbibition is impor-tant when seeds are sown into dry soils or when the contact between seedand soil is poor (Bruckler, 1983a,b; Bouaziz and Bruckler, 1989a,b) and it istherefore also likely to be variable in the seed population. Therefore, a fu-ture improvement to simulations using threshold models for prediction, invariable conditions of moisture, may be the incorporation of a suitable im-bibition model. One approach would be to apply an imbibition submodel tocover water uptake to min and water loss below min. As discussed previ-ously, imbibition of water below min can be considered a physical process.Above min the seeds become metabolically active and progress towardgermination should be predicted using a germination model. Considerationshould also be given to the incorporation of seedbed structure effects onseed-soil contact area and water uptake in the vapor phase (Hadas, 1982;

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Bruckler, 1983a,b; Bouaziz and Bruckler, 1989a,b; Wuest, Albrecht, andSkirvin, 1999).

Postgermination Seedling Growth and Emergence

There are comparatively few field studies in which the fate of nonemer-ging seedlings has been studied. In the absence of disease, most viableseeds are thought to germinate and seedling losses occur during postger-mination seedling growth (e.g., Hegarty and Royle, 1978; Durrant, 1981;Finch-Savage, Steckel, and Phelps, 1998). It is therefore necessary to con-sider and model the exhaustion of seed reserves, especially in small-seededcrops. Limiting conditions of temperature, water, and soil strength all in-crease the time to seedling emergence when reserves can be used. However,under limiting water and temperature respiration rate also decreases. Re-cent experiments have found that respiration rate in onion seedlings also de-creases as resistance to postgermination growth increases (Finch-Savageand Peach, unpublished data). In this case the total CO2 evolved for a givenincrement of seedling growth was very similar over a range of resistancesunder nonlimiting temperature and water potential. Nevertheless, seedlinggrowth, and therefore seedling emergence, can be reduced in this crop byincreased soil resistance, probably from allocation of resources to addi-tional structural components (e.g., Whalley et al., 1999).

The relative success of the model described by Finch-Savage and Phelps(1993) to describe emergence patterns implies that, once germination hasoccurred, seedling growth under a wide range of conditions does not experi-ence significant water stress even though the soil surface becomes very dry.This view is supported by the work of Vleeshouwers and Kropff (2000)who show that if the germination percentage in the soil is known, accurateprediction of numbers of seedlings emerging is possible using their modelwhich considers only temperature, soil penetration resistance, and seedweight. Few attempts have been made to include the effects of seedbedstructure (Bouaziz and Bruckler, 1989b; Mullins et al., 1996; Dürr et al.,2001), which is likely to further improve seedling growth predictions. Amodel has now been developed that includes the effect of aggregate size andorganization in the seedbed and crust development on hypocotyl growth butdoes not yet include the effects of moisture content (Dürr et al., 2001).However, reasonable predictions are possible from a simulation that ac-counts for soil moisture, temperature, soil resistance to growth, and time(Figure 2.6).

Simulations can be used to understand more about how seedling emer-gence patterns are developed. In the simple example shown (Figure 2.6),

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onion seeds were sown at the same time but at two depths in a randomizedplot experiment. Seeds that were sown more deeply germinated faster andmore uniformly (Figure 2.6e) as they were exposed to greater water poten-tials (Figure 2.6d) than those sown shallow (Figure 2.6 b and c). Soil waterpotential at the shallow sowing depth was much more variable and spenttime below b and min (Figure 2.6b), and therefore seeds germinated later(Figure 2.6c). However, the period of seedling growth was greater fromdeeper-sown seeds with a greater influence of soil impedance. The recordedemergence shows that under the conditions following this sowing, despitethe different germination times, emergence times were very similar fromshallow and more deeply sown seeds (Figure 2.6c and e). Under the drierand more variable conditions experienced at shallow sowings the predictionof seedling numbers was less accurate, underlining the difficulties de-scribed in the previous section. An additional interesting point is that sow-ing depth varies in the seed population following sowing and therefore, insimulation, seeds are assigned to different depths and characteristic basetemperatures and water potentials at random. As conditions differ in theseedbed profile, seeds may not germinate in the same set order that they areassumed to under constant laboratory conditions. For example, a faster ger-minating seed (i.e., low b) may be exposed to a lower water potential thana slow germinating seed (i.e., high b) sown deeper in the seedbed profile.Thus – b may be greater in the deeper sown, slower germinating seed,causing it to germinate faster in practice. This is accounted for in the simu-lation because the models are effectively run for each seed separately usingMonte Carlo simulation principles.

SUMMARY AND CONCLUSIONS

The complex interactions between germination and preemergence growthcharacteristics in the seed population and seedbed conditions that deter-mine seedling emergence present a challenging subject. Threshold modelscan accurately describe the range of responses from individuals within thepopulation to constant environmental conditions in the laboratory and pro-tocols are being developed to extend this to field conditions that vary. Todate there have been few attempts to use these population-based models tosimulate and predict germination and emergence in the field. The prospectslook good, but further development of the models to include greater flexi-bility to account for interactions between temperature and water potentialeffects will be required along with testing in laboratory and field conditionsthat vary. However, the difficulty in obtaining accurate seed-scale environ-mental measurements may be the factor that limits accurate predictions of

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the numbers and spread of germination and emergence time in the popula-tion. Nevertheless, accurate prediction of mean germination times and thetiming of seedling flushes seems possible.

An important application of simulation models is to understand the ap-parent contradictions that can result from field experimentation. They alsoform a powerful vehicle for the extension of scientific research to thefarmer. An immediate use for these models may be for the selection of suit-able seed sources for the site to be sown, sowing times, and suitable sites innatural populations (Battaglia, 1997). In agriculture, they could have practi-cal application, such as determining the relative timing of crop and weedpopulations to develop strategies for reducing competition. They can alsohave an educational role in developing an improved appreciation of howvariations in germination and emergence times are generated. A further useof field simulation models is to determine potential improvements ingrower practice through exhaustive scenario testing, under a wide range ofweather conditions, which is not practical by experimentation. For exam-ple, delayed emergence allows more time for seedbed deterioration andconsequential effects on the uniformity and numbers emerging from thepopulation. Delayed emergence can also result in reduced vigor and re-duced photosynthetic efficiency of individual seedlings (Tamet et al., 1996).Scenario testing can be used to develop protocols that keep seedling emer-gence time to a minimum, such as timing irrigation in relation to seeddevelopment (physiological time) rather than clock time (Finch-Savage,1990a,b). In this way, even if accurate prediction of seedling emergence un-der more extreme conditions is not possible, the models may be used toavoid these extremes by determining the timing of farmer intervention atcrucial stages to provide predictable seedling emergence.

APPENDIX

A Threshold Model for Postgermination Seedling Growth

Whalley and colleagues (1999) developed a model to describe the elonga-tion rate of a shoot that was based on the monomolecular function written as

dL

dtb A L (2.17)

where t is the thermal time, L is the shoot length, and A and b are constants.To take into account the effect of water stress and mechanical impedance onelongation rate, both A and b were scaled by the following factor:

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1 1q

qL

n

L

(2.18)

where q is the penetrometer pressure (i.e., proportional to mechanicalimpedance) and is the water stress, and qL, L, and n are all constants. qL isa conceptual value of penetrometer pressure that will just stop elongationand L, a water potential that will just stop elongation. In this form, themodel gave a reasonable description of the response of carrot and onionshoots to constant levels of mechanical impedance; however, it was poor atdescribing how shoots recovered in a stress-free environment, followingprolonged exposure to combinations of water stress and mechanical imped-ance. A practical example of this situation would be irrigation of a dry andstrong soil. To improve this aspect of the model, Whalley and colleagues(1999) allowed A to decline with thermal time according to a logistic func-tion written as

Ac

td

m1

(2.19)

where t is thermal time accumulated by a seedling and c, d, and m are con-stants. To make predictions with the model it needs to be solved numeri-cally. Some analogy can be drawn between this model for shoot elongationrate and the threshold models used to describe germination. Both modelshave a value of water potential below which no elongation can occur. In ad-dition, the shoot elongation model has an upper limit to mechanical imped-ance which stops elongation. However, the shoot elongation model is dif-ferent because the rate of elongation depends on the length of the shoot (L)and the thermal time that the seedling has accumulated (t) in addition to thedifference between the level of a physical stress (mechanical impedance, qor/and water stress, ) and its threshold value (qL and/or L). In contrast,the rate of germination at a given temperature depends only on the differ-ence between water potential and the appropriate value of base water poten-tial.

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Koller, D. and Hadas, A. (1982). Water relations in the germination of seeds. InLange. O.L., Nobel, P.S., Osmond, C.B., and Ziegler, H. (Eds.), Encyclopedia ofPlant Physiology, New Series, Volume 12B (pp. 401-431). Berlin: Springer-Verlag.

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Kruk, B.C. and Benech-Arnold, R.L. (1998). Functional and quantitative analysis ofseed thermal responses in prostrate knotweed (Polygonum aviculare) and com-mon purslane (Portulaca oleracea). Weed Science 46: 83-90.

Kruk, B.C. and Benech-Arnold, R.L. (2000). Evaluation of dormancy and germina-tion responses to temperature in Cardus acanthoides and Anagallis arvensis us-ing a screening system, and relationship with field observed emergence patterns.Seed Science Research 10: 77-88.

Labouriau, L.G. (1970). On the physiology of seed germination in Vicia gramineaSm. I. Annales Academia Brasilia Ciencia 42: 235-262.

Labouriau, L.G. and Osborn, J.H. (1984). Temperature dependence of the germina-tion of tomato seeds. Journal of Thermal Biology 9: 285-294.

Lascano, R.J. and van Babel, C.H.M. (1986). Simulation and measurement of evap-oration from bare soil. Soil Science Society of America Journal 50: 1127-1132.

Maguire, J.D. (1984). Dormancy in seeds. Advances in Research and Technology ofSeeds 9: 25-60.

Marshall, B. and Squire, G.R. (1996). Non-linearity in rate-temperature relation-ships of germination in oilseed rape. Journal of Experimental Botany 47: 1369-1375.

McDonald, M.B., Jr., Sulivan, J., and Lauer, M.J. (1994). The pathway of water up-take in maize seeds. Seed Science and Technology 22: 79-90.

McDonald, M.B., Jr., Vertucci, C.W., and Roos, E.E. (1988a). Seed coat regulationof soybean seed imbibition. Crop Science 28: 987-992.

McDonald, M.B., Jr., Vertucci, C.W., and Roos, E.E. (1988b). Soybean seed imbi-bition: Water absorption by seed parts. Crop Science 28: 993-997.

Meyer, S.E., Debaene-Gill, S.B., and Allen, P.S. (2000). Using hydrothermal timeconcepts to model seed germination response to temperature, dormancy loss, andpriming effects in Elymus elymoides. Seed Science Research 10: 213-223.

Mondal, M.F., Brewster, J.L., Morris, G.E.L., and Butler, H.A. (1986). Bulb devel-opment in onion (Allium cepa L.): I. Effects of plant density and sowing date infield conditions. Annals of Botany 58: 187-195.

Mullins, C.E., Townend, J., Mtakwa, P.W., Payne, C.A., Cowan, G., Simmonds,L.P., Daamen, C.C., Dunbabin, T., and Naylor, R.E.L. (1996). EMERGE UserGuide: A Model to Predict Crop Emergence in the Semi-Arid Tropics. Aberdeen:Department of Plant and Soil Science, University of Aberdeen.

Murdoch, A.J., Roberts, E.H., and Goedert, C.O. (1989). A model for germinationresponses to alternating temperatures. Annals of Botany 63: 97-111.

Ni, B.R. and Bradford, K.J. (1992). Quantitative models characterizing seed germi-nation responses to abscisic acid and osmoticum. Plant Physiology 98: 1057-1068.

Ni, B.R. and Bradford, K.J. (1993). Germination and dormancy of abscisic acid- andgibberellin-deficient mutant tomato seeds: Sensitivity of germination to abscisicacid, gibberellin, and water potential. Plant Physiology 101: 607-617.

Orozco-Segovia, A., González-Zertuche, L., Mendoza, A., and Orozco, S. (1996).A mathematical model that uses Gaussian distribution to analyze the germina-tion of Manfreda brachystachya (Agavaceae) in a thermogradient. PhysiologiaPlantarum 98: 431-438.

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Pemberton, M.R. and Clifford, H.T. (1994). Seed germination models. Seed Scienceand Technology 22: 209-221

Perry, D.A. (1984). Factors influencing the establishment of cereal crops. Aspects ofApplied Biology 7: 65-83.

Phelps, K. and Finch-Savage, W.E. (1997). A statistical perspective on thresholdtype models. In Ellis, R.H., Black, M., Murdoch, A.J., and Hong, T.D. (Eds.),Basic and Applied Aspects of Seed Biology (pp. 361-368). Dordrecht, the Nether-lands: Kluwer.

Probert, R.J. (2000). The role of temperature in the regulation of seed dormancy andgermination. In Fenner, M. (Ed.), Seeds: The Ecology of Regeneration in PlantCommunities, Second Edition (pp. 261-292). Wallingford, UK: CAB Interna-tional.

Richard, G. and Boiffin, J. (1990). Effets de l’état structural du lit de semences sur lagermination et la levée des cultures. In Boiffin, J. and Marin-Laflèche, A. (Eds.),La structure du sol et son évolution (pp. 112-136). Paris: Institut National de laRecherche Agronomique (INRA).

Richard, G. and Guérif, J. (1988a). Modélisation des transferts gazeux dans le lit desemence: Application au diagnostic des conditions d’hypoxie des semences debetterave sucrière (Beta vulgaris L.) pendent la germination: I. Présentation dumodèle. Agronomie 8: 539-547.

Richard, G. and Guérif, J. (1988b). Modélisation des transferts gazeux dans le lit desemence: Application au diagnostic des conditions d’hypoxie des semences debetterave sucrière (Beta vulgaris L.) pendent la germination: II. Résultats dessimulations. Agronomie 8: 639-646.

Roberts, E.H. (1988). Temperature and seed germination. In Long, S.P. and Wood-ward, F.I. (Eds.), Plants and Temperature (pp. 109-132). Cambridge, UK: Soci-ety for Experimental Biology.

Roberts, H.A. (1984). Crop and weed emergence patterns in relation to time of culti-vation and rainfall. Annals of Applied Biology 105: 263-275.

Roman, E.S., Murphy, S.D., and Swanton, C.J. (2000). Simulation of Chenopodiumalbum seedling emergence. Weed Science 48: 217-224.

Roman, E.S., Thomas, A.G., Murphy, S.D., and Swanton, C.J. (1999). Modelinggermination and seedling elongation of common lambsquarters (Chenopodiumalbum). Weed Science 47: 149-155.

Ross, H.A and Hegarty, T.W. (1979). Sensitivity of seed germination and seedlingradicle growth to moisture stress in some vegetable crop species. Annals of Bot-any 43: 241-243.

Rowse, H.R., McKee, J.M.T., and Higgs, E.C. (1999). A model of the effects of wa-ter stress on seed advancement and germination. New Phytologist 143: 273-279.

Salter, P.J. and James, J.M. (1975). The effect of plant density on the initiation,growth and maturity of curds of two cauliflower varieties. Journal of Horticul-tural Science 50: 239-248.

Schneider, A. and Renault, P. (1997). Effects of coating on seed imbibition: I.Model estimates of water transport coefficient. Crop Science 37: 1841-1849.

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Schoolfield, R.M., Sharpe, P.J.H., and Magnuson, C.E. (1981). Non-linear regres-sion of biological temperature-dependant rate models based on absolute reac-tion-rate theory. Journal of Theoretical Biology 88: 719-731.

Scott, S.J., Jones, R.A., and Williams, W.A. (1984). Review of data analysis meth-ods for seed germination. Crop Science 24: 1192-1199.

Sharpe, P.J.H. and DeMichele, D.W. (1977). Reaction kinetics of poiklotherm de-velopment. Journal of Theoretical Biology 64: 649-670.

Shrestha, A., Roman, E.S., Thomas, A.G., and Swanton, C.J. (1999). Modeling ger-mination and shoot-radicle elongation of Ambrosia artemisiifolia. Weed Science47: 557-562.

Steinmaus, S.J., Prather, T.S., and Holt, J.S. (2000). Estimation of base tempera-tures for nine weed species. Journal of Experimental Botany 51: 275-286.

Tamet, V., Boiffin, J., Durr, C., and Souty, N. (1996). Emergence and early growthof an epigeal seedling (Daucus carota L.): Influence of soil temperature, sowingdepth, soil crusting and seed weight. Soil and Tillage Research 40: 25-38.

Tarquis, A. and Bradford, K.J. (1992). Prehydration and priming treatments that ad-vance germination also increase the rate of deterioration of lettuce seed. Journalof Experimental Botany 43: 307-317.

Thornley, J.H.M. (1986). A germination model: Responses to time and temperature.Journal of Theoretical Biology 123: 481-492.

Trudgill, D.L., Squire, G.R., and Thompson, K. (2000). A thermal time basis forcomparing the germination requirments of some British herbaceous plants. NewPhytologist 145:107-114.

Vertucci, C.W. (1989). The kinetics of seed imbibition: Controlling factors and rel-evance to seedling vigor. In Stanwood, P.C. (Ed.), Seed Moisture (pp. 93-115).Special Publication No 14. Madison, WI: Crop Science Society of America

Vertucci, C.W. and Leopold, A.C. (1983). Dynamics of imbibition in soybean em-bryos. Plant Physiology 72: 190-193.

Vertucci, C.W. and Leopold, A.C. (1987). Water binding in legume seeds. PlantPhysiology 85: 224-231.

Villiers, T.A. (1972). Seed dormancy. In Kozlowski, T.T. (Ed.), Seed Biology, Vol-ume II (pp. 220-281). New York: Academic Press.

Vleeshouwers, L.M. (1997). Modeling the effect of temperature, soil penetration re-sistance, burial depth and seed weight on preemergence growth of weeds. Annalsof Botany 79: 553-563.

Vleeshouwers, L.M. and Kropff, M.J. (2000). Modeling field emergence patterns inarable weeds. New Phytologist 148: 445-457.

Wagenvoort, W.A. and Bierhuizen, J.F. (1977). Some aspects of seed germinationin vegetables: II. The effect of temperature fluctuation, depth of sowing, seedsize and cultivar, on heat sum and minimum temperature for germination.Scientia Horticulturae 6: 259-270.

Walker, A. and Barnes, A. (1981). Simulation of herbicide persistence in soil, a re-vised computer model. Pesticide Science 12: 123-132.

Wanjura, D.F., Buxton, D.R., and Stapleton, H.N. (1970). A temperature model forpredicting initial cotton emergence. Agronomy Journal 62: 741-743.

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Washitani, I. (1985). Germination-rate dependency on temperature of Geraniumcarolinianum seeds. Journal of Experimental Botany 36: 330-337.

Washitani, I. (1987) A convenient screening test system and a model for thermalgermination responses of wild plant seeds: Behavior of model and real seeds inthe system. Plant, Cell and Environment 10: 587-598.

Washitani, I. and Takenaka, A. (1984). Mathematical description of the seed germi-nation dependency on time and temperature. Plant, Cell and Environment 7:359-362.

Weaich, K., Bristow, K.L., and Cass, A. (1996). Modeling preemergent maize shootgrowth: I. Physiological temperature conditions. Agronomy Journal 88: 391-397.

Welbaum, G.E., Bradford, K.J., Yim, K.O., Booth, D.T., and Oluoch, M.O. (1998).Biophysical, physiological and biochemical processes regulating seed germina-tion. Seed Science Research 8: 161-172.

Whalley, W.R. (1993). Considerations on the use of time-domain reflectometry(TDR) for measuring soil water content. Journal of Soil Science 44: 1-9.

Whalley, W.R., Finch-Savage, W.E., Cope, R.E., Rowse, H.R., and Bird, N.R.A.(1999). The response of carrot (Daucus carota L.) and onion (Allium cepa L.)seedlings to mechanical impedance and water stress at suboptimal temperatures.Plant, Cell and Environment 22: 229-242.

Wheeler, T.R. and Ellis, R.H. (1991). Seed quality, cotyledon elongation at subop-timal temperatures, and the yield of onion. Seed Science Research 1: 57-67.

Wilson, A.M. (1973). Responses of crested wheatgrass seeds to environment. Jour-nal of Range Management 26: 43-46.

Woodstock, L.W. (1988). Seed imbibition: A critical period for successful germina-tion. Journal of Seed Technology 12: 1-15.

Wuest, S.B., Albrecht, S.L., and Skirvin, K.W. (1999). Vapor transport vs. seed-soilcontact in wheat germination. Agronomy Journal 91: 783-787.

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Chapter 3

Agronomic Factors Associated with Germination Under StressSeed and Agronomic Factors Associatedwith Germination Under Temperature

and Water Stress

Mark A. Bennett

INTRODUCTION

Substantial progress has been made in our understanding of physiologi-cal mechanisms in seeds that confer the ability to germinate under stressconditions. Parallel to this progress is a series of agronomic changes, in-cluding (1) shifts to earlier planting dates and tillage practices; (2) greaterexpectations of precision and uniformity in seedling establishment; and (3)double-cropping systems that require continued seed research and newstrategies for reliable crop production.

The objective of this chapter is to describe and review present knowledgeon physiological, morphological, and cultural factors involved in germina-tion under stress conditions. Although not intended to be a comprehensiveliterature review of this wide-ranging subject, references to related reviews,proceedings, and books are made in connection with several sections of thischapter.

The overarching goal of crop establishment is to achieve rapid and uni-form germination, followed by rapid and uniform seedling emergence plusautotrophy (Covell et al., 1986). Seeds are particularly vulnerable tostress(es) encountered between sowing and seedling establishment (Carterand Chesson, 1996). Germination and seedling establishment in crop spe-cies are the end result of a complex and interactive process, involving anumber of physiological, morphological, environmental, and cultural fac-

The assistance of Julie Hering in preparing the manuscript and Jimmie Jones in fig-ure design is appreciated. Salaries and research support provided in part by state and fed-eral funds appropriated to the Ohio Agricultural Research and Development Center, TheOhio State University.

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tors (Figure 3.1). Insights into the physiological mechanisms and culturalpractices that increase the ability of seeds to perform optimally understressful conditions will be useful for (1) sowing on atypical dates and (2)when introducing crops into new production areas or systems (Covell et al.,1986; Thiessen Martens and Entz, 2001).

SEED COATS

The seed coat, or testa, has an important role in germination under stressconditions. An intact seed coat is essential for controlled water uptake andprotection from injury to the embryo or other tissues (Chachalis and Smith,2000; Baskin and Baskin, 1998). Seeds of various Fabaceae species havebeen studied to compare traits of permeable versus water-impermeable ge-notypes. Studies with ‘Williams 82’ soybean seedlots demonstrated theability of seed coats to (1) direct water penetration to the embryo and (2)serve as a reservoir of water for the developing axis (McDonald, Vertucci,and Roos, 1988a). The testa can also decrease levels of solute leakage re-

Crop reside

Weed Seed

Microflora

Soil Structure

Soil Temperature

Water

Nutrients

Tillage System

RadicleEmergence &Root SystemDevelopment

Allelochemicals

C

B

ASeed

Pathogen Virulence

Inoculum Density

A = Germination

B = Emergence

C = Autotrophic,seedling established

Seed Size

Seed Coat

Seed Constituents(e.g., amount protein)

Seed Vigor

Seed Treatment(Chemical/Biological)

Herbicides

Herbicides

FIGURE 3.1. Schematic model of physiological, morphological, and cultural fac-tors involved in germination and seedling establishment

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sulting from seed water uptake and imbibitional damage. A comparativestudy of 19 soybean accessions with a wide range of seed size (60 to 257mg/seed) and testa color showed a testa dry weight range (5.8 to 18.3mg/seed) that was closely correlated to seed dry weight (Chachalis andSmith, 2000). Total dry weight per unit area ranged from 0.075 to 0.150mg·mm–2 and was negatively correlated with total seed dry weight. Rates ofwater uptake and testa dry weight:dry weight ratios (6.5 percent to 13.8 per-cent) were not correlated (Chachalis and Smith, 2000). Lupin seeds (espe-cially lines of Lupinus pilosus) had thinner coats when produced in a dryseason with about 50 percent of average rainfall (Miao, Fortune, andGallagher, 2001). Genetic characteristics and production environment ef-fects on seed coat structure can interact significantly.

Seed coat surface deposits, phenolic materials, and pore developmentpatterns have also been studied in relation to water uptake (Mayer andPoljakoff-Mayber, 1989). Phenolic materials in permeable and imperme-able legume seeds have not been strongly linked to water uptake patterns(Slattery, Atwell, and Kuo, 1982; Chachalis and Smith, 2001). Develop-mental studies of four soybean genotypes from maturity groups III throughV showed pores formed first around the hilum (approximately 36 days afterflowering). Pore development next encircled the seed parallel with the axisand then formed on the abaxial surface, i.e., the area covering the round faceof the cotyledon (Yaklich, Vigil, and Wergin, 1986). Soybean imbibitionstudies with four permeable and three impermeable seed lines detected alack of pores in the abaxial region of the seed coat in VLS-1, a delayed-per-meability genotype. In two lines possessing a rapid-permeability seed coatcharacteristic, pores were observed to be deep, wide open, and densely dis-tributed (Chachalis and Smith, 2001). The VLS-1 (delayed-permeability)line is black seeded; if the associated pure characteristic is inheritable,breeders could transfer this trait to yellow-seeded genotypes. Alternatively,genotypes could be selected based on pore characteristics to provide moreresistance to imbibition damage from waterlogged soil conditions, etc.

SEED SIZE

Large seed size is widely thought to improve the chances for crop emer-gence under a wide range of environments. It is also generally consideredthat, within a seedlot, seeds with a greater seed weight have greater storagereserves and thereby have increased seed vigor (Powell, 1988). Studies ofseed size effects on stand establishment are conflicting, however, and sev-eral possible explanations exist for the mixed findings. Seed size classesshould be kept distinct from seed quality (vigor) assessments. Among red

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clover seedlots, for example, the relationship between thousand seed weight(TSW) and seed vigor was weak or nonexistent (Wang and Hampton,1989). Although many reports suggest that larger seeds produce seedlingswith greater early growth and increased competitive ability against weedsand pests (Chastain, Ward, and Wysocki, 1995; Douglas, Wilkins, andChurchill, 1994; Mian and Nafziger, 1992), the sheer range of conditionsexamined in the literature is cause for careful interpretation of results.

Plants grown from smaller spring wheat (Triticum aestivum L.) seedsemerged faster but accumulated less shoot weight than plants grown fromlarge seeds (Lafond and Baker, 1986a). Seed size accounted for approxi-mately 50 percent of the variation in seedling shoot dry weight for the ninecultivars tested over two years in Saskatchewan, Canada. A large survey ofwinter wheat stand establishment in the southern Great Plains of the UnitedStates showed reduced percent emergence (45 percent) for the smallest seedclass (<19.8 g, TSW). This compared to approximately 60 percent emer-gence for seed classes of 19.8 to 22.7, 25.5 to 28.0, and >28 g per TSW(Stockton et al., 1996). Seed size studies are interesting in that the cause(s)of smaller seed can be quite diverse. Mian and Nafziger (1992) examinedthe effect of three seed sizes of soft red winter wheat, producing seed sizeclasses with sequential harvests of 21, 28, and 35 days after anthesis (DAA).All seed sizes emerged equally well in the two-year study in Illinois, eventhough the seed size range for 21 to 35 DAA lots varied considerably byyear of harvest (Mian and Nafziger, 1992). Seed size differences in otherstudies are often established by screening or other sorting procedures whichmay confound effects from (1) drought, (2) disease or insect damage, (3) earor head position, and (4) seed size and dormancy physiology interactions.

Corn seed size classes have also shown differences in imbibition, withsmall flat (SF) seeds having a faster rate of water uptake than large round(LR) kernels during initial stages of germination (Shieh and McDonald,1982). First counts of the standard germination test showed smaller seedsgerminated more rapidly than large seeds for the two inbreds tested. Similarfindings were reported by Muchena and Grogan (1977), who noted thatsmaller seeds may require less water due to less seed volume. They alsospeculate that small-seeded corn lines could provide more rapid and im-proved germination under conditions of limited soil moisture. Seed sizestudies with a sweet corn inbred divided a composite lot into LF (large flat),LR, SF, and SR (small round) classes. Under greater crop establishmentstress, the SF lot performed well in seedling dry weight accumulation (four-leaf stage) relative to the other classes (Bennett, Waters, and Curme, 1988).Seedlot nitrogen (N) concentration, TSW, and age all had significant (P <0.001) effects on perenial ryegrass (Lolium perenne L.) seed vigor (Cook-son, Rowarth, and Sedcole, 2001). N concentration accounted for more

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variability in laboratory emergence and perennial ryegrass seedling dryweight than TSW in these studies, but individual effects of N and TSW areoften confounded (Bennett, Rowarth, and Jin, 1998; Lowe and Ries, 1972).

Seed size may also be linked to emergence through soil crusts. Researchcomparing crust-tolerant and crust-susceptible sorghum genotypes indi-cated differences in seed-seedling conversion efficiency. Susceptible geno-types used about 60 percent of their initial seed weight for forming seedlingtissue, while the tolerant genotypes used only 40 percent (Soman, Jaya-chandran, and Peacock, 1992). Tolerant genotypes had longer mesocotylswith faster growth rates, allowing an avoidance mechanism for soil crustingsituations.

Carrying seed size studies to yield data comparisons is generally suspectsince many intervening factors may outweigh the original factor. The re-search by Mian and Nafziger (1992) noted a higher wheat yield in one yearfrom small seed plots, but it was largely due to reduced lodging. Many otherfactors (planting depth, planting date, optimal versus suboptimal growingconditions or cultural practices) can intervene between seed size at sowingand eventual yield. In other species, such as kura clover (Trifolium ambig-uum M. Bieb.), shoot weights may serve as a better indicator of seedlingvigor than seed size since the plant allocates a majority of its reserves to rootand rhizome development during seedling establishment (DeHaan, Ehlke,Sheaffer, 2001).

SEED WATER UPTAKE

Available soil water is an essential factor for seed germination. Uptaketypically follows a triphasic curve of (1) rapid uptake, (2) lag phase, and (3)additional hydration from cell expansion and radicle growth (Obrouchevaand Antipova, 1997). Seeds have the same general response to water supplyas to temperature fluctuations. An optimal seed substrata water status existsfor germination percentage or rates, with lower germination values on ei-ther side of the optimum (Gulliver and Heydecker, 1973; Chatterjee, Das,and Deb, 1981). Indirect effects of water status may be on (1) leaching ofendogenous inhibitors, (2) soil crusting from flooded soils followed byrapid drydown, (3) decreased oxygen availability, or (4) increased competi-tion from microbes favored by super- or suboptimal water supply.

In undamaged seed, phase I water uptake is closely linked to colloidal orphysical properties. Nitrogen and protein content are major colloidal con-stituents of many important crop species (Cardwell, 1984; Vertucci andLeopold, 1987). Seeds rich in protein imbibe more water than fat-storingseeds. Water-insoluble carbohydrates from soybean seeds were found to

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hold tenfold their weight in water, while protein held only twice its weightin water (Smith and Circle, 1972). Soybean seed parts (axes versus cotyle-dons) differed substantially in moisture content when whole seeds were al-lowed to imbibe for 72 h. Axes contained 800 g water/kg fresh weight at 48h (germination) versus 550 to 600 g water/kg fresh weight for cotyledon tis-sue with differences largely due to lipid versus carbohydrate contents (Mc-Donald, Vertucci, and Roos, 1988b). Studies with seed size of soft whitewinter wheat indicated no effect on emergence at soil water contents of 0.12to 0.16 g water/g. However, light (small) seeds, which also had the highestpercent protein content, were observed to emerge more rapidly at the lowestsoil water status (0.10 g·g–1) tested (Douglas, Wilkins, Churchill, 1994).

Seeds may become hydrated by water in the liquid or vapor phases. Un-der conditions of severe low soil water stress, seeds may be suspended instages of incomplete hydration (Chatterjee, Das, and Deb, 1982; Hegarty,1978). Metabolic changes and associated shifts in seed storage materialsmay allow water uptake to later resume or rapidly germinate upon subse-quent rainfall or irrigation (Hadas, 1982). The concept of natural priming ofseeds may also apply in these and semiarid or low water stress situations(González-Zertuche et al., 2001). Partial imbibition, inadequate for germi-nation per se, may result in a type of priming with rapid and more uniformgermination plus emergence occurring with subsequent rainfall (Kigel,1995). Seed metabolism will also vary with respect to critical hydration lev-els (Vertucci and Farrant, 1995). Exposure of perennial ryegrass and annualbluegrass seeds to controlled hydration-dehydration cycles resulted in de-layed but more uniform germination (Allen, White, and Markhart, 1993).Cycled seeds required fewer hours in contact with liquid water to germinatethan continuously hydrated seeds. A more thorough understanding of seedgermination patterns in crop species after wetting and drying cycles willbenefit seedling establishment under stressful field conditions (Koller andHadas, 1982).

Initial radicle protrusion is dependent upon cell expansion, not cell divi-sion (Haigh, 1988; Gornik et al., 1997). Minimum seed moisture contentsrequired for turgor pressure and base water potential ( b) for germinationmay vary slightly for individual seeds within a seedlot, but estimated mini-mum values for seeds of several species are reported in the literature (Table3.1). Moisture stress of –1.0 to –1.3 MPa is known to delay lettuce germina-tion (Haber and Luippold, 1960). Cultivar differences in water require-ments for tomato seed germination were noted by Liptay and Tan (1985).Using different available soil moisture (ASM) treatments of 5, 35, 60, 75,and 100 percent of a loamy sand, one cultivar germinated well at 60 percentASM or greater while the other cultivar required 100 percent ASM for opti-mal germination. Differences in seed moisture contents required for germi-

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nation have also been reported among sorghum cultivars (Mali, Varade, andMusande, 1979). In studies comparing commercial tomato (Lycopersiconesculentum) with two wild tomato species, L. chilense and Solanum pen-nellii, germination of L. esculentum appeared less sensitive to water deficits(–0.2 to –0.8 MPa) than did the two wild species (Taylor, Motes, andKirkham, 1982). Germination of all species was inhibited more by waterstress, as compared to seedling growth responses. This conclusion was alsoreached by Bhatt and Srinivasa Rao (1987) studying four L. esculentumcultivars and the wild tomato species L. pimpinellifolium. Recent work onthe genetic basis of tomato seed germination rates at reduced water poten-tial will be useful in understanding the physiological determinates of b(Foolad and Lin, 1997; Foolad et al., 1997). Base water potentials can alsochange with seed maturity. As broccoli seed matured, germination responseto osmotic stress decreased (Still, 1999). Seeds harvested at 38 days afterflowering (DAF) had a b of –0.6MPa, but the b decreased to –0.8 MPafor seeds harvested at 49 DAF, and reached an intermediate level (–0.7MPa) for 56 DAF seeds (Still, 1999).

Mucilaginous seeds are able to establish better seed-soil contact, whichassists in water uptake. Myxospermy (mucilaginous seeds) is more com-mon for seeds of plant families native to arid/semiarid regions (e.g., Brassi-caceae, Euphorbiaceae, Plantaginaceae, Labiateae) (van der Pijl, 1982). Inan attempt to mimic nature, the application of hydrophilic polymers as seedcoatings or seed furrow amendments to absorb water have given inconsis-

TABLE 3.1. Estimated minimum water potential ( min) values and base waterpotential values ( b) of various seeds. Minimum water potential is consideredthe minimum seed hydration level at which metabolic advancement will occur,and b is the threshold water potential below which germination will notproceed.

Species min (MPa) SourceTomato (Lycopersicon esculentum

Mill.)–2.45 Cheng and Bradford, 1999

Tomato –2.50 Bradford and Haigh, 1994Lettuce (Lactuca sativa L.) –2.40 Tarquis and Bradford, 1992Nemophila menziesii Aggr. –2.0 Cruden, 1974

b (MPa)

Lettuce –1.3 Haber and Luippold, 1960Wheat (Triticum aestivum L.) < –1.5 Owen, 1952Wheat < –2.0 Lindstrom, Papendick, and

Koehler, 1976

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tent results. Hydrolyzed starch-graft-polyacrylonitrile (H-SPAN) applied at2 to 5 g·kg–1 of sweet corn seed showed improved emergence, while similartests reduced cowpea (Vigna unguiculata) emergence and seedling dryweight (Baxter and Waters, 1986a). These results were likely linked to dif-ferences in seed storage materials and rates of water uptake. Laboratory ex-periments with H-SPAN on sweet corn planted into a silt loam soil at matricpotentials of –0.01, –0.40, –1.0, and –1.5 MPa were also conducted. Treatedseeds had greater imbibition, respiration, and emergence at –0.01 and –0.40MPa than control seeds, but the H-SPAN coating had a deleterious effect assoil water matric potential decreased to –1.0 and –1.5 MPa (Baxter and Wa-ters, 1986b). This effect was also reported for Russian wildrye seeds(Berdahl and Barker, 1980). For hydrophilic coatings to be most effective,soil water potential should be near field capacity initially. This water canfirst hydrate the coating sufficiently to cause germination. Coatings may beuseful in nonirrigated dryland conditions where seeds are planted just priorto or immediately after rainfall. Coatings could then trap water around theseed for improved germination.

RADICLE EMERGENCEAND ROOT SYSTEM DEVELOPMENT

Seed reserves and environmental factors largely determine the initialpatterns of germination and seminal root growth. After seed reserves are ex-hausted, however, the size and activity of the young root system plays a ma-jor role in determining the rate of early seedling shoot growth and dry mat-ter accumulation (Hoad et al., 2001). In the small grains, primary (seminal)roots develop from the radicle and comprise approximately 5 to 10 percentof the total root volume at full growth. Secondary roots (also referred to asnodal, adventitious, or crown roots) arise from nodes at the stem or tillerbase (Hoad et al., 2001). Soil compaction, greater bulk density values, highseeding rates, and moisture stress can reduce root development and seed-ling establishment. However, radicle lengths did not differ between soilcrusting-tolerant and -susceptible lines of sorghum, and no effect was de-tected of crusting treatments on radicle length (Soman, Jayachandran, andPeacock, 1992; Dexter and Hewitt, 1978).

Selection for longer pearl millet seedling root length in greenhouse sandculture correlated well with field seedling emergence and shoot height(McGrath et al., 2000). Seedlings with longer roots tolerated or avoidedmoisture stress better than five other populations (with short roots, long col-eoptile, or short coleoptiles) tested. Under flooding stress (hypoxia), corngenotypes were shown to differ in adventitious root production, with some

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hybrids producing more root weight per seedling at hypoxic levels (10.5and 14.0 KPa O2) than at ambient (20.9 KPa O2) control levels (VanToai,Fausey, and McDonald, 1988).

Critical O2 concentration (COC), or the O2 concentration below which aprocess becomes dependent on [O2], was 4.8 KPa for most of the ten corngenotypes evaluated, but 10.5 KPa for flood-susceptible genotypes Mo17and B37 (VanToai, Fausey, and McDonald, 1988). Correlations betweengermination and adventitious root formation across the ten genotypes testedwas not very high (at 2.5 KPa O2, correlations of 0.6 and 0.4 for inbreds andhybrids, respectively) (VanToai, Fausey, and McDonald, 1988). ReportedCOC values for tomato root growth (0.14 mol·m3) and barley (0.6 mol·m3

or 0.13 KPa O2) indicate the wide range among species (Benjamin andGreenway, 1979).

GENETIC LINKS TO GERMINATIONTEMPERATURE LIMITS

In temperate regions, low temperatures at the time of planting are oftenthe most limiting environmental component for germination and seedlinggrowth under early spring field conditions. Optimum temperatures for seed-ling growth and for radicle emergence (germination) are likely to differ formost species (Gulliver and Heydecker, 1973; Marsh, 1992; Nomura et al.,2001). Germination base temperatures for chickpea (0°C) and cowpea(8°C) were quite consistent, while soybean varied widely for temperate-origin genotypes (4°C) versus those with tropical origins (10°C) (Covellet al., 1986). Base temperatures for germination have also been shown to beunaffected by seed age in barley and wheat (Ellis, Hong, and Roberts, 1987;Khah, Ellis, and Roberts, 1986).

Earlier planting to achieve (1) longer growing seasons, (2) better use ofsunlight and rainfall, and (3) enhanced yield potential has placed a premiumon selecting cold-tolerant (CT) populations for major crop species (Yu andTuinstra, 2001). An example is maize, which is now grown at 55° latitude(North), despite its warm-season characteristics and subtropical origins(Shaw, 1988). As for most species examined to date, at least two mecha-nisms appear to be involved in CT of maize—one for germination andemergence and another for seedling growth (Revilla et al., 2000). For cold-tolerance breeding in tomato, Foolad and Lin (2000) suggest that each stageof plant development may require evaluation and selection. No cold-toler-ant tomato cultivar has yet been developed or released for commercial use.A more thorough understanding of CT at different growth stages in tomato(and other species) will likely require genetic mapping, cloning, and char-

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acterization of the functional genes that confer tolerance at each stage(Foolad and Lin, 2001).

An oilseed rape cultivar (Martina) showed potential to give rise to genet-ically distinct populations that could exploit different environments (Squireet al., 1997). Early germinator seeds (5°C) and viable, but nongerminatingseeds at 5°C were hand selected and selfed for testing of progeny seedlots.Germination differences were small at 19°C but large at <10°C (Squireet al., 1997). It has been noted that acceptable germplasm screening sys-tems must distinguish between temperature responses due to genotypes andresponses due to presowing environment (i.e., seed production effects onvigor) (Ellis et al., 1986). A streamlined screening protocol for faba bean(Vicia faba L.) genotype germination response to sub- and supraoptimaltemperatures was proposed by Ellis, Simon, and Covell (1987). Four tem-peratures (10°C, 20°C, 27°C, 30°C) were used with repeated probit analy-ses to compare genotypes for base and optimum temperature values. Gar-cia-Huidobro, Monteith, and Squire (1982a) used a wide range of constanttemperatures (12°C to 47°C) and showed pearl millet rate of germinationincreased linearly from a base temperature to a clearly defined optimum.Beyond the optimum, germination rate decreased linearly with increasedtemperature to a Tmax and no germination.

Use of alternating or fluctuating temperatures may be more appropriatefor predicting or interpreting field germination and emergence data. Be-yond the seed dormancy breaks associated with alternating temperature re-gimes, fluctuating temperatures are thought to (1) increase the maximumfraction that will germinate in a seed population and (2) possibly increasethe rate of germination (Garcia-Huidobro, Monteith, and Squire, 1982b). Instudies with pearl millet, large diurnal temperature amplitudes (8°C) andtemperatures from 15° to < 42°C accelerated the germination rate. Differ-ences were small relative to comparable average constant temperatures butmay be important for seeds germinating in field environments.

SEED PRODUCTION AND SEED VIGOR

Among the many cultural factors and management decisions that impactgermination under environmental stress, an important early indicator ofcrop establishment is seedlot vigor. A high quality or vigorous seedlot pos-sesses the ability to germinate and emerge uniformly and quickly under thewide range of conditions (temperature, moisture, biotic stresses, etc.) com-monly encountered in field settings (Association of Official Seed Analysts[AOSA], 1983). Environmental conditions during seed development canhave major effects on seed quality (Wulff, 1995; Syankwilimba, Cochrane,

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and Duffus, 1997). Seed production per se will not be covered extensivelyin this section, but implications for better germination under stressful con-ditions will be addressed.

Seed development and seed production of Brassica species is a usefulstarting point for examining the development of seed vigor. Red cabbageand rapeseed were selected from among the brassica crops for studies con-ducted by Still and Bradford (1998) and Still (1999). The indeterminategrowth pattern and extended flowering period (typically 35 d) of brassicasforce seed producers into a compromise over mature seed yield potentialversus shattering losses.

Rapeseed maximum seed dry weight was achieved by 33 days after flow-ering, while for red cabbage this stage was reached by 54 DAF (Still andBradford, 1998). Different sensitivities to water stress (reduced water po-tential) (Still and Bradford, 1998) and exogenous abscisic acid (ABA) treat-ments (Benech-Arnold, Fenner, and Edwards, 1991) were linked to the en-vironment experienced by the mother plant during seed development.Temperature thresholds may also be linked to different seed maturity stages(Ellis et al., 1986).

For many species and seed crops, it is difficult to determine when physi-ological maturity (PM) or maximum seed quality has occurred and whetherthis developmental stage is synchronized with maximum seed dry weight.In rapeseed, PM was reached 4 to 9 d after maximum seed dry weight; in redcabbage, PM was observed at 6 or 7 d later than maximum dry weight (Stilland Bradford, 1998). Best seed quality (PM) is reported to occur after maxi-mum seed dry weight accumulation for many other crops, including sweetcorn (Wilson and Trawatha, 1991), barley (Pieta-Filho and Ellis, 1991), andPhaseolus vulgaris (Sanhewe and Ellis, 1996). Seed moisture can also be auseful indicator of seed quality development in field bean, with seed qualityassessment (standard germination, controlled deterioration, and conductiv-ity) values leveling off at about 0.4 g water/g fresh weight. This seed mois-ture content did not differ across years, anthesis dates, or pod locations onthe plant (Coste, Ney, and Crozat, 2001).

Shattering is not a critical factor in sh2 sweet corn seed production, butslow drydown in the field has lead to studies on earlier harvests (0.45 to0.65 g water/g fresh weight) for this unique endosperm type (Borowski,Fritz, and Waters, 1991). Sweet corn seed can be harvested at higher thannormal (0.35 to 0.45 g water/g fresh weight) moisture levels with proper at-tention to harvesting, handling, and drying operations (Borowski, Fritz, andWaters, 1995). Continued research on membrane and pericarp integritychanges during seed production will help to provide flexibility in seed har-vest windows and supply good yields of high-quality seed.

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An early indicator of loss of seed vigor is a narrowing of the range ofconditions (e.g., temperatures, water) in which seeds will germinate (Ab-dul-Baki and Anderson, 1972). New seedling imaging systems for quick as-sessment of seed vigor will be useful in seed production and seed inventorydecisions (Sako et al., 2001).

SOWING DEPTHS AND PLANTER TECHNOLOGY

Seedling establishment is often compromised by wide-ranging soil mois-ture conditions (near field capacity to levels too dry for germination), plant-ing depths (<1 cm to 15 cm or more), and seedbed temperatures (0.25°C to0.5°C). Delays in germination and emergence subjects seedlings to greaterrisk from soil crusting impedance and greater competition or damage fromvarious pathogens, insects, and weeds. Deep planting (8 to 10 cm or more)is often required to place seeds of barley, winter wheat, and other crops inmoist soil (Lindstrom, Papendick, and Koehler, 1976; Radford, 1987).

It is useful, especially in deep planting situations, to separately considerthe processes of (1) germination and radicle emergence and (2) subsurfaceseedling elongation. The emergence phase (subsurface coleoptile elonga-tion, etc.) is generally more sensitive to marginal seedbed conditions (Lind-strom, Papendick, and Koehler, 1976). Long coleoptile length (usuallyhighly correlated with seed weight) is clearly desirable when deep sowingis required in crop production. Within most barley, oat, and wheat cultivars,larger seed with good germinability produced longer coleoptiles (Kaufman,1968). The extra seed reserves for emergence in larger seed plus longer col-eoptiles were both linked to more successful seedling establishment. In stud-ies with seven barley cultivars, constant temperatures of <10°C and >20°Creduced coleoptile lengths for all genotypes (Radford, 1987). At 10°C, col-eoptile length ranged from 64 to 106 mm, while at 25°C the lengths droppedto 58 to 80 mm. Optimal barley seed zone temperatures varied by cultivar.One line showed optimal coleoptile growth at 10°C, 10 or 15°C, and 15 or20°C, while four cultivars produced optimal coleoptiles anywhere acrossthe 10 to 20°C range (Radford, 1987). It is recommended in deep-sowingsituations that furrows be formed over the seed rows to minimize the actualdepth of soil covering, e.g., deep sow at 110 mm and firm soil directly abovethe seed with a press wheel to leave 75 to 80 mm of soil actually over theseed. (Radford, 1987).

Precision agriculture techniques may also be useful for sowing depth andvariable seed placement decisions. Within-field variability leads to substan-tial ranges of soil temperatures and moisture, and refinements in planter en-gineering show promise for dealing with these key variables (Carter and

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Chesson, 1996; Price and Gaultney, 1993). The use of global positioningsystems (GPS) and geographic information systems (GIS) allow the map-ping of fields for many applications, including seed placement, for im-proved stand establishment. Field studies with several shrunken-2 sweetcorn cultivars showed that seedling emergence for an entire field wasgreater using variable planting depths (2 to 4 cm) based on mapped soil typedifferences versus a single planting depth of 2 cm (Barr, Bennett, andCardina, 2000). Additional mapping data on soil compaction (Hakansson,Voorhees, and Riley, 1988; Wolfe et al., 1995) and a better understanding ofcultivar interactions will improve the accuracy and utility of precisionplanting techniques for a wider range of crop species and field environ-ments.

TILLAGE SYSTEMS AND SOIL STRUCTURE EFFECTS

Worldwide concerns about soil erosion and deteriorating soil structurehave spurred research and use of various conservation tillage systems thatpreserve more crop residue at or near the soil surface. Germination andemergence can be impacted by increased residues in many ways, including(1) cooler, wetter microclimates, (2) decreased seed-soil contact for wateruptake, (3) allelochemical interactions, and (4) modified levels of ethyleneproduction and removal (Douglas, Wilkins, Churchill, 1994; Creamer,Bennett, and Stinner, 1996; Hadas and Russo, 1974a,b; Karssen and Hil-horst, 1992; Chase, Nair, and Putnam, 1991; Arshad and Frankenberger,1990). Many crop producers also feel pressured to plant earlier in order tomeet market windows or optimize light interception, and earlier plantingsare often made into cold, wet soils regardless of the tillage system employed(Hakansson, Voorhees, and Riley, 1988). Soil compaction is commonlycaused by vehicle traffic on wet soil, which puts additional stress on germi-nation and seedling emergence. Systems or environments that slow standestablishment also prolong the period of seedling vulnerability to soil im-pedance, diseases, insects, and weed competition (Wolfe et al., 1995;Mohler and Galford, 1997).

Soil attributes and critical threshold values for a number of variableshave recently been proposed by Pilatti and deOrellana (2000) for mollisolsin Argentina. Among the many attributes considered, at least four are linkedto germination and seedling establishment concerns. They are (1) root pen-etration resistance/impedance, (2) surface crusting potential, (3) water stor-age capacity, and (4) total biological activity. The effort to describe criticalvalues of an “ideal soil” and establish threshold values for 25 or more attrib-utes seems promising for more accurate assessment and crop decision-mak-

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ing processes. Combining this information with GPS/GIS precision farm-ing systems (Barr, Bennett, and Cardina, 2000) should aid in overcomingmany crop establishment obstacles in coming decades.

Genotypes of important crop species, including corn, are known to differin their germination and seedling growth response to low oxygen concen-trations (VanToai, Faussey, and McDonald, 1988). While O2 content maybe more closely linked to soil drainage systems, soil type, and topographythan to tillage systems, higher crop residues can also slow the loss of water.In the study by VanToai, Faussey, and McDonald (1988), only high-vigorlots of inbred and hybrid corn lines were assessed to avoid any confoundingof seed vigor with hypoxia or anoxia responses. Low O2 levels are usuallymore limiting during germination than after radicle protrusion, which likelyfacilitates at least some increase in O2 availability (Al-Ani et al., 1985;Wuebker, Mullen, and Koehler, 2001). Fluctuating from high (content of20.0 KPa O2) to low O2 concentration was most damaging to corn germina-tion and seedling growth, especially when a period of true anoxia wasimposed (VanToai, Faussey, and McDonald, 1988). In species which are ex-tremely tolerant to flooding, such as rice (Oryza sativa L.) and barn-yardgrass (Echinochloa crus-galli L.), low O2 actually stimulates coleoptilegrowth while inhibiting root development (Rumpho et al., 1984; Alpi andBeevers, 1983). For corn, moderate levels of hypoxia (10 to 14 KPa O2) alsostimulated shoot growth of the five hybrids tested, but not the inbreds(VanToai, Faussey, and McDonald, 1988).

The occurrence of ethylene (C2H4) in soils is also important due to itsmany effects on plant development, from seed germination to senescence(Raven, Evert, and Eichhorn, 1997). Changes in levels of organic matterand associated soil microorganisms with various tillage and soil manage-ment systems can be expected to affect ethylene production, removal, andstability (Arshad and Frankenberger, 1990). The biologically active rhizo-sphere and spermosphere are likely to be very active sites for C2H4 genera-tion and consumption, with possible effects on crop and weed seed germi-nation plus seedling establishment (Karssen and Hilhorst, 1992).

INTERACTIONS WITH SEED TREATMENTSAND OTHER CROP PROTECTION CHEMICALS

Fungicide and insecticide seed treatments are often employed to protectcrops from biotic stress. Emergence from cold (2 to 7°C), wet soils is oftenslow and incomplete, with stand establishment appearing to differ for vari-ous seed treatments (Smiley, Patterson, and Shelton, 1996). Changes in till-age operations (e.g., increased use of conservation tillage or stubble-mulch

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systems) have led to more research or optimal treatments for these modifiedmicroenvironments (Bradley et al., 2001). Planting depth can also affect therecommended treatments, with some products not suggested for seedingsmade deeper than 5 cm. Three greenhouse and seven field experiments wereconducted with deeply planted winter wheat to compare the efficacy of fiveseed fungicide products (Smiley, Patterson, and Rhinhart, 1996). In thegreenhouse studies, treated seed was planted 2.5 cm deep into moist (7, 10,or 15 percent water) and warm (24°C) silt loam soil, then topped off with 10cm of dry soil to simulate planting 12.5 cm deep into a stubble-mulch fal-low system. Field study plantings were at 2.5 to 12.7 cm deep into warmsoils (21 to 27°C at seed zone) with seed zone water contents of 5 to 17 per-cent. Three of the seed fungicide treatments evaluated had variable effectson seedling emergence or established stand density values (Smiley, Patter-son, and Rhinhart, 1996). Coleoptile lengths were not affected by the fungi-cide treatment. Seed fungicide treatment decisions can interact with (1)planting depth, (2) irrigation availability, (3) planting season and likelihoodof soil crusting, (4) species or class (i.e., hard-red versus soft-white wheat),and (5) key pathogens associated with given fields or planting season. Natu-ral resistance to various diseases and pests has been linked to colored (pig-mented) seedcoats. Red pericarps have been linked to grain mold resistancein sorghum (Esele, Frederiksen, and Miller, 1993). It is also believed thatgeneral resistance to pathogens is associated with phytoalexin (pigment)accumulation in sorghum plant tissues in response to pathogen infection(Nicholson et al., 1987). Recent work by Pedersen and Toy (2001) testedthe combined effects of plant and seed color on sorghum germination,emergence, and other agronomic factors. Using 20 near-isogenic lines,seedling emergence was higher for red-seed versus white-seed phenotypes(Pedersen and Toy, 2001). Grain sorghum markets, however, often preferwhite grain, which is free of pigment stains. Purple plant phenotypes pro-duced seed with (1) higher cold germination and accelerated aging valuesand (2) greater seedling elongation at 10 d versus results from tan pheno-types, although standard (warm) germination values were not different(Pedersen and Toy, 2001). Higher grain yields were associated with whiteseeded, purple plant types.

Unexpected losses in seedling establishment (and eventual yields) canalso occur from crop responses to multiple pesticide applications. Interac-tions among fungicides, insecticides, and herbicides can be complicatedfurther by soil characteristics (Morton et al., 1993). Soil moisture, pH lev-els, and organic matter content may all influence the actual amount ofchemical taken up by a young plant. For example, if a systemic soil-appliedinsecticide such as terbufos is taken up in greater than normal amounts anddistributed at high levels throughout the young plant, its presence can re-

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duce the metabolism of later herbicide applications (Morton et al., 1993).Herbicide rates that are normally safe and free of phytotoxic effects canthen cause foliar injury and stand losses. Cold stress, seedling size, and en-dosperm class were also shown to influence sweet corn response to fourherbicide treatments in field and controlled environment studies (Bennettand Gorski, 1989). Introduction of new crop protection chemistry and newgermplasm call for careful compatibility studies, especially for seedling es-tablishment in stress environments.

SCREENING PROTOCOLS FOR GERMINATION TOLERANCETO LOW TEMPERATURE AND WATER STRESS

The study of germination stress tolerance in field settings is difficult.Soil temperature and moisture ranges needed for careful cultivar or germ-plasm evaluations are often lacking or are unpredictable (Schell et al., 1991;Blacklow, 1972; Washitani, 1987). Many researchers and crop practitionershave noted that use of controlled environment settings would be a more effi-cient strategy for examining genotype differences in germination and seed-ling emergence (Heydecker and Coolbear, 1977; Khan, 1992; Tadmor,Cohen, and Harpaz, 1969; McGrath et al., 2000).

Changes in alfalfa (Medicago sativa L.) emergence and seedling heightafter laboratory selection at suboptimal temperatures (<10°C) successfullyimproved seedling heights in the field for some populations without chang-ing other agronomic and forage quality traits.

Seedling height appeared to be a better trait than germination time onwhich to base predicted field performance if traits are measured in lab orgreenhouse studies (Klos and Brummer, 2000). The consistence of labora-tory and field responses to recurrent selection varied considerably withinthe six alfalfa cultivars assessed. A field location and population interactionwas also observed for seedling height, due to both rank and magnitude dif-ferences (Klos and Brummer, 2000). Future evaluations of response to se-lection for such traits should therefore be performed at multiple locations.

It has also been noted that at suboptimal temperature ranges for a givencrop, thermal time and germination of different individuals and fractions ofthe seed lot (population) are normally distributed. Less variation is ob-served when supraoptimal temperatures are imposed (Covell et al., 1986;Ellis, Simon, and Covell, 1987). Screening procedures for selecting grainlegume germplasm (chickpea, lentil, cowpea, soybean) tolerant to suboptimaltemperatures, based on cumulative germination and thermal time patterns,are well described by Covell and colleagues (1986). It is also useful to dis-tinguish between (1) genotype × temperature responses and (2) presowing

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or seed production environment effects linked to temperature responses if atruly acceptable germplasm screening protocol is desired (Ellis et al.,1986).

Higher catalase activity, lower lipoperoxidation, higher total oxygenconsumption at 3°C, and a doubling of fructan content were all correlatedwith the improved cold tolerance of oat cultivar OT220 versus the cold-sen-sitive cultivar America (Massardo, Corcuera, and Alberdi, 2000). Oxygen-scavenging enzymes, such as catalase, provide one mechanism for reducingoxidative injury due to cold stress. Lipoperoxidation in ‘America’ oat em-bryos increased 25 percent when germinated at 30°C versus 17°C, whilelipoperoxidation did not increase with cold treatment of the cold-tolerantcultivar OT220 (Massardo, Corcuera, and Alberdi, 2000). These and otherphysiological responses to cold described previously are correlative evi-dence that may be important links to genetic differences. These responsesalso have potential use in broader germplasm screening programs for ger-mination tolerance to low temperatures. Embryo adenosine triphosphate(ATP) levels of two corn hybrids imbibed for 64 h were different at 10°C butnot at 20°C (Schell et al., 1991). Cold test germination, emergence index,field emergence and dry weight (30 days after planting) values showedgood agreement with embryo ATP levels for these hybrids. Schell and col-leagues (1991) observed that imbibition times of 16 h may be used if ATPaccumulation rates, rather than ATP content/embryo values, are analyzed.

Lafond and Baker (1986b) assessed the germination responses of ninespring wheat cultivars to varying levels of temperature and moisture stress.Temperature ranges (5 to 30°C) and moisture stress using polyethylene gly-col (PEG8000) solutions with osmotic potentials of 0.0, –0.4, and –0.8 MPa(at 10°C and 20°C) gave final germination values of over 90 percent for allenvironments tested. Consistent cultivar rankings and differences (althoughmagnitude decreased) were reported across the range of 5 to 30°C. In-creasing the (osmotic) water stress from –0.0 to –0.8 MPa caused mediangermination time to increase from 90 h to 156 h at 10°C, and from 36 h to 64h at 20°C. Relative ranking of germination times for the nine wheatcultivars was consistent over the levels of moisture stress. Seed and seedlingtolerance to soil moisture stress is another important trait to test, but it gen-erally receives less attention than low-temperature tolerance (Hegarty,1977; Bradford, 1995). Various systems for controlled water stress havebeen used for seed germination and priming studies (Pavmar and Moore,1968; Bennett and Waters, 1984; Bradford, 1997) and are again more reli-able than using a range of field experiments. Water potential has also beenshown to affect the temperature range over which optimal germination wasobserved (Sharma, 1976; Kebreab and Murdoch, 2000). Optimal germina-tion of Orobanche aegyptiaca at 0.0 MPa occurred over 17 to 26°C (9°C

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range) compared with 17 to 20°C (3°C range) at –1.25 MPa. Optimum ger-mination temperature for this parasitic weed also tended to decrease withdecreasing water potential (Kebreab and Murdoch, 2000), and these pointsshould be considered if combining temperature and water stress assess-ments (Gummerson, 1986). Drought-tolerance assessments used for wholeplants may also hold promise for screening seeds and seedlings (Ali Dibet al., 1994; Bajji, Lutts, and Kinet, 2001).

Germination of sugarbeet (Beta vulgaris L.) seed submerged in hydro-gen peroxide and water has recently been proposed for screening cultivarand seedlot vigor (McGrath et al., 2000). Thirty-nine commercial seedlotsrepresenting 24 cultivars were tested in a range of laboratory and field ex-periments. Total germination (96 h) in 0.3 percent H2O2 was identified asthe best laboratory screen. McGrath and colleagues (2000) observe that al-though it is unlikely a water germination test can be developed to fullymimic field conditions, it should be useful in evaluating relative emergencepotential for species that tolerate immersion for several days. Physiologicaland agronomic information from the germination tests will be used to iden-tify target genes for use as markers in breeding for improved field emer-gence.

CONCLUDING REMARKS

Crop physiology and management studies often describe and quantifythe changes plant breeders and geneticists have delivered in new germplasmbut rarely address the specific changes needed to advance crop establish-ment, yield potential, or other agronomic goals (Snape, 2001). As discussedthroughout this chapter, germination and seedling establishment in the fieldis a complex process influenced by many interacting factors. Advances ingenetics and genomics will contribute much precision to the next wave ofcrop physiology and seedling establishment research. Extreme environ-mental stresses will always pose limitations for crop establishment, butcontinued progress in germplasm screening protocols and crop manage-ment research should also lead to new varieties with a tailored set of agro-nomic practices for given environments and cultural practices.

REFERENCES

Abdul-Baki, A.A. and Anderson, J.D. (1972). Physiological and biochemical dete-rioration of seeds. In Kozlowski, T.T. (Ed.), Seed Biology, Volume II (pp. 283-315). New York: Academic Press.

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Al-Ani, A., Bruzan, F., Raymond, P., Saint-Ges, V., Leblanc, J.M., and Pradet, A.(1985). Germination, respiration and adenylate energy charge of seeds at variousoxygen partial pressures. Plant Physiology 79: 885-890.

Ali Dib, T., Monneveux, P., Acevedo, E., and Naeliot, M.M. (1994). Evaluation ofproline analysis and chlorophyll fluorescence quenching experiments as droughttolerance indicators in durum wheat (Triticum turgidum L. var. durum). Euphyt-ica 79: 65-73.

Allen, P.S., White, D.B., and Markhart, A.H. (1993). Germination of perennialryegrass (Lolium perenne) and annual bluegrass (Poa annua) seeds subjected tohydration-dehydration cycles. Crop Science 33: 1020-1025.

Alpi, A. and Beevers, H. (1983). Effects of oxygen concentration on rice seedlings.Plant Physiology 71: 30-34.

Arshad, M. and Frankenberger, W.T. Jr. (1990). Ethylene accumulation in soil in re-sponse to organic amendments. Soil Science Society of America Journal 54:1026-1031.

Association of Official Seed Analysts (1983). Seed Vigor Testing Handbook.AOSA Handbook 32. Lincoln, NE: Association of Official Seed Analysts.

Bajji, M., Lutts, S., and Kinet, J.M. (2001). Water deficit effects on solute contribu-tion to osmotic adjustment as a function of leaf ageing in three durum wheat(Triticum durum Desf.) cultivars performing differently in arid conditions. PlantScience 160: 669-681.

Barr, A., Bennett, M., and Cardina, J. (2000). Geographic information systemsshow impact of field placement of sh2 sweet corn stand establishment. Hort-Technology 10: 341-350.

Baskin, C.C. and Baskin, J.M. (1998). Seeds: Ecology, Biogeography and Evolutionof Dormancy and Germination. San Diego, CA: Academic Press.

Baxter, L. and Waters, L., Jr. (1986a). Effect of a hydrophilic polymer seed coatingon the field performance of sweet corn and cowpea. Journal of the American So-ciety of Horticultural Science 111: 31-34.

Baxter, L. and Waters, L., Jr. (1986b). Effect of a hydrophilic polymer seed coatingon the imbibition, respiration and germination of sweet corn at four matric po-tentials. Journal of the American Society of Horticultural Science 111: 517-520.

Benech-Arnold, R.L., Fenner, M., and Edwards, P.J. (1991). Changes in germin-ability, ABA content and ABA embryonic sensitivity in developing seeds of Sor-ghum bicolor (L.) Moench. induced by water stress during grain filling. NewPhytologist 118: 339-347.

Benjamin, L.R. and Greenway, H. (1979). Effects of a range of oxygen concentra-tion on porosity of barley roots and on their sugar and protein concentrations.Annals of Botany 43: 383-391.

Bennett, J.S., Rowarth, J.S., and Jin, Q.F. (1998). Seed nitrogen and potassium ni-trate influence browntop (Agrostis capillaris L.) and perennial ryegass (Loliumperenne L.) vigour. Journal of Applied Seed Production 16: 77-81.

Bennett, M.A. and Gorski, S.F. (1989). Response of sweet corn (Zea mays) endo-sperm mutants to chloracetamide and thiocarbamate herbicides. Weed Technol-ogy 3: 475-478.

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Bennett, M.A. and Waters, L., Jr. (1984). Influence of seed moisture on lima beanstand establishment and growth. Journal of the American Society of Horticul-tural Science 109: 623-626.

Bennett, M.A., Waters, L., Jr., and Curme, J.H. (1988). Kernel maturity, seed size,and seed hydration effects on the seed quality of a sweet corn inbred. Journal ofthe American Society of Horticultural Science 113: 348-353.

Berdahl, J.D. and Barker, R.E. (1980). Germination and emergence of Russian wildrye seeds coated with hydrophilic materials. Agronomy Journal 72: 1006-1008.

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Borowski, A.M., Fritz, V.A., and Waters, L., Jr. (1991). Seed maturity influencesgermination and vigor of two shrunken-2 sweet corn hybrids. Journal of theAmerican Society of Horticultural Science 116: 401-404.

Borowski, A.M., Fritz, V.A., and Waters, L., Jr. (1995). Seed maturity and desicca-tion affect carbohydrate composition and leachate conductivity in sh2 sweetcorn. HortScience 30: 1396-1399.

Bradford, K.J. (1995). Water relations in seed germination. In Kigel, J. and Galili,G. (Eds.), Seed Development and Germination (pp. 351-396). New York: Mar-cel Dekker, Inc.

Bradford, K.J. (1997). The hydrotime concept in seed germination and dormancy.In Ellis, R.H., Black, M., Murdoch, A. J., and Hong, T.D. (Eds.), Basic and Ap-plied Aspects of Seed Biology (pp. 349-360). Dordrecht, the Netherlands: KluwerAcademic Publishers.

Bradford, K.J. and Haigh, A.M. (1994). Relationship between accumulated hydro-thermal time during seed priming and subsequent seed germination rates. SeedScience Research 4: 63-69.

Bradley, C.A., Wax, L.M., Ebelhar, S.A., Bollero, G.A., and Pedersen, W.L. (2001).The effect of fungicide seed protectants, seeding rates, and reduced rates of her-bicides on no-till soybean. Crop Protection 20: 615-622.

Cardwell, V.B. (1984). Seed germination and crop production. In Tesar, M.B. (Ed.),Physiological Basis of Crop Growth and Development (pp. 53-91). Madison,WI: American Society of Agronomy.

Carter, L.M. and Chesson, J.H. (1996). Two USDA researchers develop a moisture-seeking attachment for crop seeders that is designed to help growers plant seed insoil sufficiently moist for germination. Seed World 134 (March): 14-15.

Chachalis, D. and Smith, M.L. (2000). Imbibition behavior of soybean [Glycinemax (L.) Merrill] accessions with different testa characteristics. Seed Scienceand Technology 28: 321-331.

Chachalis, D. and Smith, M.L. (2001). Seed coat regulation of water uptake duringimbibition in soybeans [Glycine max (L.) Merr.]. Seed Science and Technology29: 401-412.

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Chase, W.P., Nair, M.G., and Putnam, A.R. (1991). 2,2'-1,1'-azobenzene: Selectivetoxicity of rye (Secale cereale L.) allelochemicals to weed and crop species. II.Journal of Chemical Ecology 17: 9-19.

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Chatterjee, D., Das, D.K., and Deb, A.R. (1981). Water uptake and diffusivities ofgerminating gram, cotton, soybean and cowpea seeds. Seed Research 9: 109-221.

Chatterjee, D., Das, D.K., and Deb, A.R. (1982). Water absorption, diffusivity andseed-surface soil water matric potentials of germinating maize seeds. Seed Re-search 10: 46-52.

Cheng, Z. and Bradford, K.J. (1999). Hydrothermal time analysis of tomato seedgermination responses to priming treatments. Journal of Experimental Botany50: 89-99.

Cookson, W.R., Rowarth, J.S., and Sedcole, J.R. (2001). Seed vigor in perennialryegrass (Lolium perenne L.): Effect and cause. Seed Science and Technology29: 255-270.

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Covell, S., Ellis, R.H., Roberts, E.H., and Summerfield, R.J. (1986). The influenceof temperature on seed germination rate in grain legumes: I. A comparison ofchickpea, lentil, soybean and cowpea at constant temperatures. Journal of Ex-perimental Botany 37: 705-715.

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Ellis, R.H., Covell, S., Roberts, E.H., and Summerfield, R.J. (1986). The influenceof temperature on seed germination rate in grain legumes: II. Intraspecific varia-tion in chickpea (Cicer arietinum L.) at constant temperatures. Journal of Exper-imental Botany 37: 1503-1515.

Ellis, R.H., Hong, T.D., and Roberts, E.H. (1987). Comparison of cumulative ger-mination and rate of germination of dormant and aged barley seed lots at differ-ent constant temperatures. Seed Science and Technology 15: 717-727.

Ellis, R.H., Simon, G., and Covell, S. (1987). The influence of temperature on seedgermination rate in grain legumes: III. A comparison of five faba bean genotypes

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at constant temperatures using a new screening method. Journal of ExperimentalBotany 38: 1033-1043.

Esele, J.P., Frederiksen, R.A., and Miller, F.R. (1993). The association of genescontrolling caryopsis traits with grain mold resistance in sorghum. Phytopath-ology 83: 490-495.

Foolad, M.R. and Lin, G.Y. (1997). Genetic potential for salt tolerance during ger-mination in Lycopersicon species. HortScience 32: 296-300.

Foolad, M.R. and Lin, G.Y. (2000). Relationship between cold tolerance duringseed gemination and vegetative growth in tomato: Germplasm evaluation. Jour-nal of the Amererican Society of Horticultural Science 125: 679-683.

Foolad, M.R. and Lin, G.Y. (2001). Relationship between cold tolerance duringseed gemination and vegetative growth in tomato: Analysis of response and cor-related response to selection. Journal of the American Society of HorticulturalScience 126: 216-220.

Foolad, M.R., Stoltz, T., Dervinis, C., Rodriquez, R.L., and Jones, R.A. (1997).Mapping QTLs conferring salt tolerance during germination in tomato by selec-tive genotyping. Molecular Breeding 3: 269-277.

Garcia-Huidobro, J., Monteith, J.L., and Squire, G.R. (1982a). Time, temperatureand germination of pearl millet (Pennisetum typhoides S. and H.): I. Constanttemperatures. Journal of Experimental Botany 33: 288-296.

Garcia-Huidobro, J., Monteith, J.L., and Squire, G.R. (1982b). Time, temperatureand germination of pearl millet (Pennisetum typhoides S. and H.): II. Alternatingtemperature. Journal of Experimental Botany 33: 297-302.

González-Zertuche, L., Vázquez-Yanes, C., Gamboa, A., Sánchez-Coronado, M.E.,Aguilera, P., and Orozco-Segovia, A. (2001). Natural priming of Wigandiaurens seeds during burial: Effects on germination, growth and protein expres-sion. Seed Science Research 11: 27-34.

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Hadas, A. and Russo, D. (1974a). Water uptake by seeds as affected by water stress,capillary conductivity, and seed-soil water contact: I. Experimental study. Agron-omy Journal 66: 643-647.

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Hadas, A. and Russo, D. (1974b). Water uptake by seeds as affected by water stress,capillary conductivity, and seed-soil water contact: II. Analysis of experimentaldata. Agronomy Journal 66: 647-652.

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Hegarty, T.W. (1978). The physiology of seed hydration and dehydration, and therelation between water stress and the control of germination: A review. Plant,Cell and Environment 1: 101-119.

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Khan, A.A. (1992). Preplant physiological seed conditioning. Horticultural Review14: 131-181.

Kigel, J. (1995). Seed germination in arid and semiarid regions. In Kigel, J. andGalili, G. (Eds.), Seed Development and Germination (pp. 645-649). New York:Marcel Dekker, Inc.

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Lafond, G.P. and Baker, R.J. (1986a). Effects of genotype and seed size on speed ofemergence and seedling vigor in nine spring wheat cultivars. Crop Science 26:341-346.

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Lafond, G.P. and Baker, R.J. (1986b). Effects of temperature, moisture stress, andseed size on germination of nine spring wheat cultivars. Crop Science 26: 563-567.

Lindstrom, M.J., Papendick, R.I., and Koehler, F.E. (1976). A model to predict win-ter wheat emergence as affected by soil temperature, water potential, and depthof planting. Agronomy Journal 68: 137-141.

Liptay, A. and Tan, C.S. (1985). Effect of various levels of available water on ger-mination of polyethylene glycol (PEG) pretreated or untreated tomato seeds.Journal of the American Society of Horticultural Science 110: 748-751.

Lowe, L.B. and Ries, S.K. (1972). Effects of environment on the relation betweenseed protein and seedling vigor in wheat. Canadian Journal of Plant Science 52:157-164.

Mali, C.V., Varade, S.B., and Musande, V.G. (1979). Water absorption of germinat-ing seeds of sorghum varieties at different moisture potentials. Indian Journal ofAgricultural Science 49: 22-25.

Marsh, L. (1992). Emergence and seedling growth of okra genotypes at low temper-atures. HortScience 27: 1310-1312.

Massardo, F., Corcuera, L., and Alberdi, M. (2000). Embryo physiological re-sponses to cold by two cultivars of oat during germination. Crop Science 40:1694-1701.

Mayer, A.M. and Poljakoff-Mayber, A. (1989). The Germination of Seeds. Oxford,UK: Pergamon Press.

McDonald, M.B., Jr., Vertucci, C.W., and Roos, E.E. (1988a). Seed coat regulationof soybean seed imbibition. Crop Science 28: 987-992.

McDonald, M.B., Jr., Vertucci, C.W., and Roos, E.E. (1988b). Soybean seed imbi-bition: Water absorption by seed parts. Crop Science 29: 993-997.

McGrath, J.M., Derrico, C.A., Morales, M., Copeland, L.O., and Christenson, D.R.(2000). Germination of sugar beet (Beta vulgaris L.) seed submerged in hydro-gen peroxide and water as a means to discriminate cultivar and seedlot vigor.Seed Science and Technology 28: 607-620.

Mian, A.R. and Nafziger, E.D. (1992). Seed size effects on emergence, head num-ber, and grain yield of winter wheat. Joural of Production and Agriculture 5:265-268.

Miao, Z.H., Fortune, J.A., and Gallagher, J. (2001). Anatomical structure and nutri-tive value of lupin seed coats. Australian Journal of Agricultural Research 52:985-993.

Mohler, C.C. and Galford, A.E. (1997). Weed seedling emergence and seed sur-vival: Separating the effects of seed position and soil modification by tillage.Weed Research 37: 147-155.

Morton, C.A., Harvey, R.G., Kells, J.J., Landis, D.A., Lueschen, W.E., and Fritz,V.A. (1993). In-furrow terbufos reduces field and sweet corn (Zea mays) toler-ance to nicosulfuron. Weed Technology 7: 934-939.

Muchena, S.C. and Grogan, C.O. (1977). Effect of seed size on germination of corn(Zea mays L.) under simulated water stress conditions. Canadian Journal ofPlant Science 57: 921-923.

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Nicholson, R.L., Kollipara, S.S., Vincent, J.R., Lyons, P.C., and Cadena-Gomez, G.(1987). Phytoalexin synthesis by the sorghum mesocotyl in response to infectionby pathogenic and nonpathogenic fungi. Proceedings of the National Academyof Sciences 84: 5520-5524.

Nomura, K., Endo, I., Tateishi, A., Inoue, H., and Yoneda, K. (2001). A chilling-insensitive stage in germination of a low-temperature-adapted radish, rat’s tailradish (Raphanus sativus L.) cv. “Pakki-hood.” Scientia Horticulturae 90: 209-218.

Obroucheva, N.V. and Antipova, O.V. (1997). Physiology of the initiation of seedgermination. Russian Journal of Plant Physiology 44: 250-264.

Owen, P.C. (1952). The relation of germination of wheat to water potential. Journalof Experimental Botany 3: 188-203.

Pavmar, M.T. and Moore, R.P. (1968). Carbowax 6000, mannitol, and sodium chlo-ride for simulating drought conditions in germination studies of corn (Zea maysL.) of strong and weak vigor. Agronomy Journal 60: 192-195.

Pedersen, J.F. and Toy, J.J. (2001). Germination, emergence and yield of 20 plant-color, seed-color near-isogenic lines of grain sorghum. Crop Science 41: 107-110.

Pieta-Filho, C.P. and Ellis, R.H. (1991). The development of seed quality in springbarley in four environments: II. Field emergence and seedling size. Seed ScienceResearch 1: 179-185.

Pilatti, M.A. and deOrellana, J.A. (2000). The ideal soil: II. Critical values of an“ideal soil,” for mollisols in the north of the Pampean region in Argentina. Jour-nal of Sustainable Agriculture 17: 89-111.

Powell, A.A. (1988). Seed vigour and field establishment. Advances in Researchand Technology of Seeds 16: 419-426.

Price, R.R. and Gaultney, L.D. (1993). Soil moisture sensor for predicting seedplanting depth. Transactions of the American Society of Agricultural Engineers36: 1703-1711.

Radford, B.J. (1987). Effect of cultivar and temperature on the coleoptile length andestablishment of barley. Australian Journal of Experimental Agriculture 27:313-316.

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Revilla, P., Malvar, R.A., Cartea, M.E., Butrón, A., and Ordás, A. (2000). Inheri-tance of cold tolerance at emergence and during early season growth in maize.Crop Science 40: 1579-1585.

Rumpho, M.E., Pradet, A., Khalik, A., and Kennedy, R.A. (1984). Energy chargeand emergence of the coleoptile and radicle at varying oxygen levels in Echin-ocloa crus-galli. Physiologia Plantarum 62: 133-138.

Sako, Y., McDonald, M.B., Fujimura, K., Evans, A.F., and Bennett, M.A. (2001).A system for automated seed vigor assessment. Seed Science and Technology29: 625-629.

Sanhewe, A.J. and Ellis, R.H. (1996). Seed development and maturation in Phase-olus vulgaris: II. Post-harvest longevity in air-dry storage. Journal of Experi-mental Botany 47: 959-965.

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Schell, L.P., Danehower, D.A., Anderson, J.R., Jr., and Patterson, R.P. (1991).Rapid isolation and measurement of adenosine triphosphate levels in corn em-bryos germinated at suboptimal temperatures. Crop Science 31: 425-430.

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Shieh, W.J. and McDonald, M.B., Jr. (1982). The influence of seed size, shape andtreatment on inbred seed corn quality. Seed Science and Technology 10: 307-313.

Slattery, H.D., Atwell, B.J., and Kuo, J. (1982). Relationship between colour, phe-nolic content and impermeability in the seed coat of various Trifolium sub-terraneum L. genotypes. Annals of Botany 50: 373-378.

Smiley, R.W., Patterson, L.M., and Rhinhart, K.E.L. (1996a). Fungicide seed treat-ment effects on emergence of deeply planted winter wheat. Journal of Produc-tion and Agriculture 9: 564-570.

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Smith, A.K. and Circle, S.J. (1972). Chemical composition of the seed. In Smith,A.K. and Circle, J.J. (Eds.), Soybeans: Chemistry and Technology (pp. 61-93).Westport, CT: AVI Publ. Co.

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Syankwilimba, I.S.K., Cochrane, M.P., and Duffus, C.M. (1997). Effect of elevatedtemperatures during grain development on seed quality of barley (Hordeumvulgare L.). In Ellis, R., Black, M., Murdoch, A., and Hong, T. (Eds.), Basic andApplied Aspects of Seed Biology (pp. 585-592). Dordrecht, the Netherlands:Kluwer Academic Publishers.

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Tadmor, N.H., Cohen, Y., and Harpaz, Y. (1969). Interactive effects of temperatureand osmotic potential on the germination of range plants. Crop Science 9: 771-773.

Tarquis, A. and Bradford, K.J. (1992). Prehydration and priming treatments that ad-vance germination also increase the rate of deterioration in lettuce seed. Journalof Experimental Botany 43: 307-317.

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Vertucci, C.W. and Farrant, J.M. (1995). Acquisition and loss of desiccation toler-ance. In Kigel, J. and Galili, G. (Eds.), Seed Development and Germination(pp. 237-271). New York: Marcel Dekker, Inc.

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Washitani, I. (1987). A convenient screening test system and a model for thermalgermination responses of wild plant seeds: Behaviour of model and real seeds inthe system. Plant, Cell and Environment 10: 587-598.

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Chapter 4

Methods to Improve Seed Performance in the FieldMethods to Improve Seed Performancein the Field

Peter Halmer

INTRODUCTION

A wide range of techniques is now used to help sow seeds and to improveor protect seedling establishment and growth under the changeable environ-ments and seedbed constraints reviewed in Chapters 1 and 3. These tech-niques constitute the postharvest processing necessary to prepare seed forsowing and optional treatments that are generally described in the industryand scientific literature as “seed enhancements” or “seed treatments.”

In the first comprehensive review of this subject, Heydecker and Cool-bear (1977) distinguished the purposes of seed treatment as follows: to se-lect, improve hygiene and mechanical properties, break dormancy, advanceand synchronize germination, apply nutrients, and impart stress tolerance.Subsequent reviewers (e.g., Taylor et al., 1998) adopted similar schemes.Halmer (2000), for example, grouped practical seed treatment technologiesinto operational categories in the following way:

• Conditioning or processing—by cleaning, purification, and fraction-ation, using mainly mechanical techniques such as size and densitygrading, polishing, scarification, and color sorting

• Protection—by applying active ingredients, usually synthetic fungi-cides and insecticides (The agrochemical industry commonly callsthis technology “seed treatment,” using the term in a narrower sense.)

• Physiological enhancement or “seed invigoration”—by hydrationtechniques such as priming, or applying active substances such asplant growth regulators, to exploit the ability of most species to inter-rupt the germination process by drying, and to resume the processwhen seeds are reimbibed, without vital harm (Some authors restrictthe expression “seed enhancement” specifically to describe thesetechniques.)

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• Coating—by pelleting or encrusting, to alter handling or imbi-bitional characteristics or to carry pesticides, micronutrients, andbeneficial microorganisms

The focus of this chapter is on the last two categories—in the main to re-view progress in seed coating and, especially, in physiological enhance-ment. These technologies, which some call “functional” seed treatments,are mainly used at present for high-value crops in intensive agriculture butin the future may have wider applications.

This review continues by considering the underlying mechanisms ofphysiological seed enhancement, previously evaluated by Bray (1995) andMcDonald (2000). Recent research in the disciplines of molecular and cellbiology, biophysics, and the modeling of germination is giving conceptualinsights into these processes, which may provide convenient methods forfurther improvement of seed quality. In particular, attention has been ad-dressed to identifying biochemical, biophysical, and morphological mark-ers that can be used to dissect key germination processes. Study areas mostdirectly relevant to an appreciation of physiological enhancement include(1) the mechanisms of cell and embryo expansion; (2) the preparation forcell division; (3) endosperm weakening by hydrolases; and (4) the mecha-nisms of desiccation tolerance, including protection of the state of cyto-plasm and membranes and maintenance of DNA structure during drying,air-dry storage, reimbibition, and germination. A chapter such as this canextract only the main threads from the large quantity of detailed material onthese topics, and attention will be directed to key reviews at appropriatepoints.

CHANGING SEED FORM AND LOT COMPOSITION

Sorting

Conventional processing technology includes sorting and grading seeds—exploiting superficial external seed features such as size, shape, color, sur-face texture, density, and buoyancy in air. Seed quality is purified and lotsare “upgraded” by removing contaminants, and seed that is outside specifi-cations is rejected. To supplement these well-known techniques, novel seedselection and sorting principles have been developed in recent years to re-move fractions that have higher proportions of weak or dead seeds. Simak(1984) used water flotation to separate dead and viable forest plant seedsthat had been previously imbibed and dried to amplify their density differ-ence. Aqueous buoyancy sorting can also be effective after priming, e.g., by

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discarding low-density fractions before osmoprimed tomato and lettuceseeds are dried (Taylor et al., 1992). Taylor, McCarthy, and Chirco (1982)devised mixtures of polar organic solvents (chloroform and hexane) to sep-arate dry seed batches by density. Equipment has been engineered to handlethese solvents in a way that is safe for the seed and the operator, and thissorting technique is now used commercially for high-value horticulturaland ornamental seeds. Jalink and colleagues (1998) recently developed aninnovative variation on color sorting, using laser-induced fluorescence todetect the residual content of chlorophyll in seed coats, which in some casesis undetectable to the human eye. It is thought that the amount of the pig-ment is inversely related to the maturity of the seed. This technique appearsto have practical value for upgrading tomato, pepper, leek, cucumber, andcabbage seed lots, and equipment is becoming commercially available tocarry out this patented technique. Seeds are fed past a photoelectric cell,which triggers a jet of air to remove colored individuals one at a time. In thefuture, X-radiography might offer another real-time sorting principle, usingdecision logic to identify normal and anomalous embryo structures, e.g., intomato seeds which develop an internal free space after priming andredrying (Downie, Gurusinghe, and Bradford, 1999).

Planting

Precision Sowing Systems

Many horticultural field root and salad crops and ornamental productionsystems are based on crop uniformity and must be precision sown in definedpatterns to optimize yield and harvest quality. These crops are precisionsown either directly where they are grown or are raised as seedlings in pro-tected conditions—either in nursery beds or in soil blocks, paper pots, flator plug trays in growing media—for later transplantation into pots or thefield, or into phenolic foam cubes for hydroponic propagation. In contrast,plant spacing is not usually a critical factor in arable, grass, and cover crops,which are sown by broadcasting or drilling in rows in or onto bare soil, orare “direct seeded” directly into existing pastures, turf, or crop stubble.

The natural shape of many seed species is not ideal for precision seeddrills, even after size grading. Also, although designed to cope with dry anddusty field conditions, most drills are vulnerable to blockage by misshapenseeds or dust, and seed flow can be impeded by sticky or rough seed sur-faces. In these situations, coating and pelleting are valuable seed enhance-ments to improve the accuracy of mechanical singulation. Modern preci-sion drills have three main designs. In the cell-feed system, seeds are

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collected in deep holes in the rim of a rotating metal wheel, into which theymust fit completely, before being prised out by an ejector plate at the outletpoint. In the belt-feed method, a small endless rubber belt with one to threerows of holes punched in it conveys seeds to the exit point from the seederunit. In the vacuum-feed (or pneumatic or “air planter”) system, suction isapplied to one side of a rotating disc perforated with lines of regularlyspaced holes, onto which the seeds are pulled and transported to the dis-charge point where a blanking plate cuts off the vacuum. Belt and vacuumseed drills are used not only for planting many types of horticultural speciesbut, increasingly, for planting large-seeded crops such as maize, sunflower,cotton, soybeans, and beans. The vacuum-seeding principle is also fre-quently used to sow tray formats, e.g., using nozzle arrays or flat templateplates drilled with holes to suit the layout. Nursery beds are sown using fielddrills or simpler perforated drum seeders. Grain drills have much simplerdesigns, e.g., with seed being carried by a fluted or studded feed roller toflexible tubes for delivery to the ground.

Coating: Pelleting, Encrusting, and Film Coating

The primary historical purposes of pelleting and encrusting is to build upseed to change its shape, weight, size, or surface structure, by applying vari-able amounts of filler materials and binders—typically to make seeds fitdrills better. Pelleting is usually carried out to make irregularly shaped seedovoid and smooth, or to make small seeds much larger. In comparison, seedcoating (“minipelleting” or “encrusting”) applies less material, so that theoriginal seed shape is still more or less visible. Apart from improving drillperformance, coating is also used to upgrade size ranges and to increaseweight to prevent drift, e.g., for aerial seeding of range and amenity grasses.Pelleted and coated seed is commonly colored to make it easier to find seedsafter drilling and check depth and spacing, as well as to identify companies,varieties, or treatments. Species pelleted in substantial commercial amountsinclude sugar beet (quantitatively by far the largest use), carrot, celery, chic-ory and endive, leek, lettuce, onion, pepper, tomato, and to a lesser extentsome Brassica species and “super-sweet” corn varieties, and certain flowerspecies, particularly those with tiny seeds. There are potential applicationsfor seed coating in crops that do not need precision sowing, e.g., to reducesize variation of maize and sunflower kept in inventory, which are typicallysold in up to six different size grades. Pelleting and coating can be used tocarry nutrients and growth-stimulating materials, including plant growthregulators (PGRs).

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Thin film coating is employed mainly to apply colorants and pesticidetreatments onto seeds, in a firmer and more uniform way than can beachieved using conventional slurry application techniques. As well as im-proving treatment accuracy, film coating is used to minimize chemical dust-off losses during seed handling and drilling, and exposure of the operatorswho handle treated seed. It also presents seed for sale in a cosmetically at-tractive form. Characteristically, each seed is covered with a water-perme-able polymer layer, which adds about 1 to 10 percent to the weight so thatshape and size are little changed. Film-coating techniques are now well es-tablished for many high-value horticultural seed species and are beingadopted for treating some higher-volume crops, such as maize, sunflower,canola, alfalfa, clover, and some grasses. Film coating is also widely used toapply insecticides and fungicides to the outside of pelleted seed: in somecases this is a preferred method of application to minimize phytotoxic ef-fects, especially where these treatments have to be applied at very highloading rates.

The treatment of seeds with agrochemical formulations is now a marketof huge worldwide value and importance that is steadily growing as alterna-tives to application by sprays or granules become available. Though mostseed sown is treated in this way, this is not the place to cover the subject.Brandl (2001) has summarized recent progress, including the developmentin recent years of “active ingredients” with systemic modes of action thatcan protect plants for several months into the life of the crop. It is worthmentioning here in passing, however, that a number of active ingredientshave moderate side effects on seed performance, by slowing germination orproducing seedling abnormalities by imposing phytotoxic stresses; suchdefects are regarded as acceptable, commercially, considering the protec-tion benefits delivered to the crop.

Commercial film coating, pelleting, and coating systems are often run assecret processes, and there are very few detailed investigations of this sub-ject in the scientific literature, although patents give useful descriptions andinsights into the technologies involved. Halmer (2000) has reviewed equip-ment and techniques and the general types of filler materials and binders inpelleting, encrusting, and film coating, and the processes for applying pesti-cide formulations onto seeds using these and other techniques.

Coating to Modify Imbibition and Germination

Usually and understandably, commercial pelleting, coating, and film-coating types are designed to impose minimal mechanical or physiologicalbarriers on germination while reshaping and resizing seed strongly enough

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for drilling purposes and for the adhesion of protective treatments. How-ever, the materials used can also be tailored to modify seed water availabil-ity and gaseous exchange, and so control the timing of germination andemergence.

Several studies have been published on ideas to use treatment or film-coating techniques to manipulate seed imbibition characteristics. Hydro-phobic materials may be included within or around the pellet or coatingfabric to allow seeds to germinate under wet conditions, for species such asonion where that can be erratic, or filler materials may be incorporated togive a more porous structure to the matrix. Some pellet types are designedto disintegrate rapidly or split after imbibition to expose the seed. Severalstudies over the years have investigated the promotion of emergence by in-cluding calcium or magnesium peroxide in the pellet to supply more oxy-gen in waterlogged environments (see Ollerenshaw, 1988).

Various film-coating polymers (e.g., Schmolka, 1988; Taylor et al.,1992; Ni, 2001) have been evaluated as potential barrier layers to alleviateseed imbibitional chilling injury leading to poor seedling establishment invulnerable crops, such as certain cultivars of large-seeded grain legumesand super-sweet corn, especially when seed coat layers are abnormally thinor damaged. Damage can involve disruption of oil bodies and membranes,and the leakage of cell contents from the outermost embryo tissues, includ-ing the solutes measured in the electrical conductivity test for these species,and may lead to the death of cells on the cotyledon surface (see references inChachalis and Smith, 2000). Seed coats of many species are not as vulnera-ble to rapid imbibition, due in part to the presence of semipermeable layersin the seed coat tissues, which restrict solute diffusion and leakage rates (seeTaylor et al., 1998). Humidification is another approach to alleviate theimbibitional chilling injury problem—simply by raising seed moisture con-tent in a damp atmosphere for several days (Taylor et al., 1992).

An extension of this concept is to delay imbibition with water-resistantpolymers until climatic conditions become suitable for continued cropgrowth (e.g., Watts and Schreiber, 1974). In recent years this type of tech-nology has attracted a great deal of commercial interest, though develop-ments have mainly been relayed through the trade press and very little hasbeen published yet in the scientific literature detailing mechanisms andfield performance. Polymers with in vitro temperature-dependent water-permeability properties have been advocated to coat seeds for early plantingso that they can imbibe only when favorable moisture and temperature con-ditions have developed (Stewart, 1992); among the applications being eval-uated are the coordination of the flowering of parental lines planted at thesame time for hybrid maize seed production. For a somewhat similar pur-pose, another water-resistant polymer coating has been evaluated as a tool

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to give a wider window of opportunity for sowing canola in the autumn innorthern American latitudes, just before soils freeze over winter, for earlieremergence and crop maturation and greater yields compared to the normalspring-seeding time. These technologies are potentially powerful but willhave to perform very reliably in changeable soil environments, particularlyif they are to be used in space-planted crops that do not have a compensa-tory growth habit.

By contrast, the inclusion of water-attracting materials can aid imbibi-tion and give more intimate seed-soil contact, or may retain moisture in thevicinity of the seed as soils dry. For example, some success has been re-ported using nonionic surfactants (Aksenova et al., 1994) and hydrophilicgels (Henderson and Hensley, 1987). The starch-based or polyacrylate/polyacrylamide polymers used commercially as soil amendments to retainwater in agricultural and horticultural situations are also advocated to treatseeds. Such superabsorbent materials need to be applied and kept dryenough to prevent the seed batch congealing into an unusable mass.

Other Planting Systems

Hydroseeding and seed tapes. Some seed is sown using specialist tech-niques that do not involve conventional drills or coating. In hydroseedingaqueous slurries of seed and other materials are sprayed to enable fast andconvenient seeding of amenity areas or steep slopes with grass, wildflowers,or other groundcover vegetation. The patent literature contains quite a fewvariations on the seed tape format, in which seed is stuck or embedded ran-domly or in patterns between layers of biodegradable paper or plastic, etc.,in porous mats, grids, or narrow strips, some incorporating growing media,which are laid out dry in the ground (e.g., Holloway, 1999; Meikle andSmith, 2000). These sowing systems can help suppress weed growth andare suitable for placement of much higher doses of nutrients, moisture re-tention agents, and protective chemicals than could be directly coated ontoseeds without encountering phytotoxic effects.

Pregermination. The slurry and tape-sowing concepts have each beendeveloped for planting pregerminated hydrated seeds. Fluid drilling, thebest-known technique of this type, is used in some places to establish small-seeded vegetables, e.g., celery and tomato. Seed is allowed to complete ger-mination in an aerated medium of relatively high water potential and, afterremoval of ungerminated individuals by density separation, the sproutedseeds are suspended in a viscous gel and precision sown by extrusion intothe soil (Pill, 1991; Far, Upadhyaya, and Shafii, 1994). Low water poten-tials or leachable plant growth inhibitors, such as abscisic acid, can be used

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to synchronize the germination process (Finch-Savage and McQuistan,1989). A novel propagation concept proposes the use of seed tapes to raiseand transplant germinated seedlings or more fully developed transplants inmoist “paper pockets” containing hygroscopic material, etc. (Ahm, 2000).The success of such propagation systems relies in part on arranging timelyseedling production and optimized seedbed conditions to ensure develop-ment of the young seedlings with minimal desiccation damage, and they arebest suited to situations in which production follows a fixed plan and is notlikely to be interrupted by bad weather.

In another type of pregermination treatment, development is suspendedjust after radicle emergence and seed is dried to produce high-viability lotsfor conventional sowing. In a patented technique that is marketed at presentmainly for flower species, fully imbibed seeds are germinated to the pointwhere radicles are just visible, sorted by machine vision, flotation, or othermeans to remove ungerminated seeds, and dried to induce desiccation toler-ance. This can produce either damp pregerminated seed (30 to 55 percentmoisture content) with a shelf life of a few weeks at ambient temperature, ordry seed that is viable for a few months (Bruggink and van der Toorn, 1995,1996). McDonald (2000) reports that dehydration of pregerminated seedsusing cool low relative humidity air (e.g., 11 percent and 35 percent RH, at5ºC) successfully imposes desiccation tolerance and extends storage life upto four weeks. When circumstances allow, it is also possible to use undriedfreshly primed seed. Alternatively, Sluis (1987) patented the idea of pro-ducing a moist pellet from primed seed incorporating materials such asosmotica or abscisic acid to slow germination; at refrigerated temperaturesand/or under reduced atmospheric pressure the seed microenvironmentwould be sufficiently stabilized to allow several weeks of storage life.

PHYSIOLOGICAL ENHANCEMENT

Priming and Related Hydration Techniques

Germination enhancement technologies based on presowing seed hydra-tion have attracted considerable interest in both seed physiological researchand industry circles, where they have been extensively commercialized.Heydecker’s work (Heydecker, Higgins, and Turner, 1975) is often taken asthe starting point for modern research in this area, and a substantial litera-ture has developed since. By manipulating water relations to exploit mostseeds’ natural ability to survive one or more cycles of imbibition and dry-ing, subsequent germination is made faster and often more uniform—whichHeydecker distinguished as the “advancement” and “priming” responses,

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respectively. In recent years, however, the meaning of the term priming hasevolved from its original specific sense of increased germination synchronyand is now commonly used to describe seed presowing hydration methodol-ogies without discrimination, where seeds are imbibed by whatever means(e.g., Khan, 1992; Parera and Cantliffe, 1994b; Taylor et al., 1998; McDon-ald, 2000).

The hydration treatments regulate the germination process by manipu-lating temperature, seed moisture content, and duration. Water is eithermade freely available to the seed (as in steeping or soaking) or restricted to apredetermined moisture content or a programmed sequence of moisturecontents, typically using water potentials between –0.5 MPa and –2.0 MPa.Some positively photoblastic species benefit from treatment under light ofappropriate wavelengths, and it is possible to include other materials suchas nutrients and growth regulators with the water. Then, usually, seeds aredried back prior to further treatment as required, e.g., by coating or treat-ment with pesticides, for storage and sowing.

One practical drawback is that primed seeds often, but not always, deteri-orate faster during storage and accelerated aging than untreated seeds.Symptoms include the onset of a reducing rate, uniformity, and final level ofgermination, and an increase in the proportion of abnormal seedlings—although the degree of the problem in susceptible species varies amongseed lots and with the extent of priming performed and storage conditions.A related problem is the increasing injury seen as priming is allowed to pro-ceed too far, reflecting the familiar fact that seeds’ ability to survive dryingand the dry state for extended periods of time is progressively lost as germi-nation progresses. It is important to know how to optimize the priming foran individual seed lot. What applies to one lot need not necessarily apply toanother; indeed, differences between lots can be more important than dif-ferences between cultivars.

Typical responses to priming are faster and closer spread of times to ger-mination and emergence over all seedbed environments and wider tempera-ture range for germination, leading to better crop stands, and hence im-proved yield and harvest quality, especially under suboptimal and stressgrowing conditions in the field, though responses can vary due to fluctuat-ing water availability and temperature. Times to reach 50 percent of maxi-mum emergence (T50) can typically be decreased by up to one-third underenvironmental conditions in seedling production practice. Seed lots differin the magnitude of their response to a standard priming treatment; in gen-eral, slower-germinating lots exhibit the greatest benefit (Brocklehurst andDearman, 1983; Bradford, Steiner, and Trawatha, 1990).

Primed seeds are now used commercially in the production of manyhigh-value crops where reliably uniform germination is important, such as

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the field seeding or plug production of leek, tomato, pepper, onion, and car-rot, and the production of potted or bedding ornamental herbaceous plants,such as cyclamen, begonia, pansy (Viola sp.), Polyanthus sp., and primrose(Primula sp.), and several culinary herbs, as well as for some large-volumefield crops, such as sugar beet and turf grasses. Due to its ability to raise theupper temperature limit for germination, priming is also very valuable forcircumventing the secondary thermodormancy that results when imbibedseeds are likely to be exposed to supraoptimal temperatures for too long,e.g., in susceptible cultivars of lettuce, celery, and pansy (Hill, 1999; Mc-Donald, 2000).

Technologies

Fundamentally, three strategies are used to deliver and restrict the amountof water and to supply air: submersion in solutions of osmotica in water,mixing with moist solid particulate materials, and hydration with wateronly. Though new descriptive names have proliferated in recent years, thebasic principles of almost the entire array of priming technologies used inresearch and the seed industry today were mapped out in Heydecker andCoolbear’s (1977) insightful review. Seed companies usually perform com-mercial priming treatments, using proprietary methodologies and systemsthat handle quantities ranging from tens of grams to several tonnes at a time,and that are often kept secret.

Priming protocols for a “new” species have been developed mainly on anexperimental basis. There is a substantial research literature reporting andcomparing priming approaches and conditions for many species: Welbaum,Shen, and colleagues (1998b) and McDonald (2000) provide selected bibli-ographies of priming techniques that have been used successfully on a widerange of crops. A rough rule of thumb is to start by using the temperaturesconsidered optimal for germination of untreated seeds, water potentialsequal to or less than the threshold water potential at which emergence of theembryonic axis (usually the radicle) is prevented, and durations from one tothree weeks, but these conditions may not prove optimal for priming. Someseeds benefit from prewashing before priming to remove germination in-hibitors, e.g., sugar beet and umbelliferous species.

Because of the variability in response between seed lots, optimum prim-ing conditions—choosing the balance between the most rapid germination,and the longest storage life—often need in practice to be determined on acase-by-case basis for many species (Welbaum, Shen, et al., 1998). Con-ducting pilot priming runs on small samples achieves this empirically, i.e.,by varying somewhat the final water potential and perhaps the stages taken

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to reach it, their duration and (less commonly) temperature, and testing ger-mination responses.

A continuing goal for the seed industry is to find simple means to deter-mine these parameters quickly and reliably, to complement or replace exist-ing test procedures. Therefore, considerable research interest is directed to-ward identifying marker signals that correlate well with the degree ofadvancement and/or the loss of desiccation tolerance in individual seed lots.These indicators might provide means to assess the potential effectivenessof priming a seed lot, to help set operating parameters before the treatmentis started, and to monitor its progress in real time prior to radicle emergence.They might also provide research tools to develop and distinguish newpriming approaches and protocols (Job et al., 2000). The seed merchant andthe grower would also value post facto tests that give a measure of whether,and how well, a seed lot has been physiologically enhanced, and to predictits storage life. Such tests should ideally give precise and reliable informa-tion across all varieties and seed lots and should also be fast and convenientto perform, in an industry in which many seed lots are processed on a just-in-time basis and decisions are needed quickly.

Osmopriming. Osmotic priming of seeds (also known as osmopriming orosmoconditioning) describes contacting seeds with aerated solutions of lowwater potential, usually by submersion, which are rinsed off afterward. Thisis still regarded by many researchers to be the standard priming technique,and treatment on the surface of paper or other fibers moistened with solu-tion or immersed in small continuously aerated columns continues to becommon study method in which only small quantities of seed are required.

Mannitol or inorganic salts [such as KH2PO4, KH(PO4)2, K3PO4, KCl,KNO3, Ca(NO3)2, and various mixtures of these] have been used exten-sively as osmotica but, because of their low molecular size, these are capa-ble of being absorbed by the seeds, which has been associated with toxicside effects in some cases. Na salts, however, which tend to be more toxic tosome common agricultural seeds than K salts, have been proposed to inducetolerance to saline conditions, e.g., in tomato (Cano et al., 1991). Hey-decker, Higgins, and Turner (1975) first suggested the alternative of usingmoderately high molecular weight fractions of polyethylene glycol (PEG,most commonly, 6 to 8 kDa), whose large size precludes it from entering theseed, and this is now widely used as a preferred osmoticum by many in re-search and the seed industry.

Care must be taken, particularly when using viscous PEG solutions, toensure adequate gas exchange by constant vigorous aeration or by usingstirred bioreactors (Bujalski et al., 1991). Some seeds, particularly onions,are reported to only osmoprime satisfactorily using air enriched with oxy-gen (Bujalski and Nienow, 1991). A patented process using a semiper-

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meable membrane to separate seeds from an osmoticum contained in theouter jacket of a rotating tube has been devised for osmopriming small seedquantities, such as small-seeded flower species, and for seeds with mucilag-inous coats that can cause difficulties in other priming methods (Rowse andMcKee, 1999).

Matrix priming. Solid matrix priming (matripriming and the closely al-lied matriconditioning technique) mixes seed with solid insoluble matrixparticles, such as exfoliated vermiculite, diatomaceous earth, or cross-linked highly water-absorbent polymers, and water in predetermined pro-portions (Taylor, Klein, and Whitlow, 1988; Eastin, 1990; Tsujimoto, Sato,and Matsushita, 1999). Seeds slowly imbibe to reach an equilibrium hydra-tion level, determined by the reduced matrix potential of the water adsorbedon the particle surfaces, and after the incubation the moist solid material isremoved by sieving. It is important in this approach to ensure adequate aer-ation and prevent the formation of temperature gradients in the seed mass,and that the surplus matrix material can be removed without mechanicallydamaging the seed or leaving too much dust on it.

Hydropriming. Hydropriming is currently used both in the sense of thecontinuous or staged addition of a limited amount of water (e.g., van Pijlenet al., 1996; McDonald, 2000) and also the sense of imbibition in water for ashort period (e.g., Gurusinghe and Bradford, 2001) with or without subse-quent incubation in humid air (Fujikura et al., 1993).

Slow imbibition is the basis of the patented drum priming and related ex-perimental techniques (Rowse, 1996; Warren and Bennett, 1997), whichevenly and slowly hydrate seeds up to a predetermined moisture content—typically about 25 to 30 percent on a fresh-weight basis—by misting, con-densation, or dribbling. Tumbling in a rotating cylinder ensures that seedlots are evenly hydrated, aerated, and temperature controlled during thedamp incubation stage. De Boer and Boukens (1999) have devised a prim-ing system using direct hydration from a humid atmosphere (RH > 98 per-cent) to control the final stage of imbibition and maintain the moisture con-tent in a static seed mass. These approaches have the practical economicadvantages that the production of waste materials associated with osmo-priming or matripriming is avoided, and that the relatively modest amountsof water involved are removed by drying.

Submerged aerated hydration, very akin to steeping, has been proposedas a treatment to enhance the germination of horticultural Brassicas (Thorn-ton and Powell, 1992). Davidson (1981) proposed seed steeping in high ox-ygen atmospheres.

Steeping. Hydropriming for longer periods followed by drying back tothe original seed moisture content is also commonly known as steeping.Steeping treatments, e.g., at up to 30°C for several hours (sometimes the du-

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ration needs to be adjusted for individual seed lots), are now widely per-formed to remove residual amounts of germination inhibitors and/or to in-filtrate chemical fungicide treatments to control deep-seated seed-bornediseases, such as for sugar beet and umbelliferous species (Maude, 1996).

At its very simplest, on-farm steeping and sowing of wet seed has a longhistory. This was done where circumstances allowed in the days before themechanisation of sowing, and similar overnight steeping is even now advo-cated as a pragmatic, low cost, and low risk agricultural method for enhanc-ing crop establishment in developing countries, e.g., for groundnut, maize,upland rice, and chickpea crops (Massawe et al., 1999; Harris et al., 1999).Direct benefits are reported to include improved drought tolerance, earlierflowering, and higher seed/grain yield. Rice is also steeped in some mecha-nized farm situations, in part just to increase seed weight to aid in sowingfrom the air.

Other methods. The older research literature has a few reports (e.g., seeHeydecker and Coolbear, 1977; Hegarty, 1978) of the benefits of seed hard-ening (two to three cycles of steeping and drying), particularly for droughttolerance, but the subject has not received much research attention of lateand these approaches do not seem to be in wide commercial use, possiblybecause they are cumbersome to perform.

Changes in Endogenous Microflora

Seed-borne microflora, including pathogens, can increase during prim-ing but cannot necessarily be fully controlled by conventional fungicidesincluded in the osmopriming solutions alone (Maude et al., 1992; Nasci-mento and West, 1998), and seeds may need subsequent treatment after dry-ing. However Petch and colleagues (1991) concluded that the presence oflarge number of microorganisms did not greatly affect seed performancewhen the same PEG osmoticum was used three times with leek and twicewith carrot seed.

Wet Heat Treatments to Eradicate Seed-Borne Disease

Short hot water treatments are used to disinfect seeds, typically at tem-peratures of about 50° to 60°C for up to about 30 minutes for some small-seeded species such as flowers, and, for example, very brief exposure tosteam is being evaluated as an organic treatment for cereal seed (Maude,1996; Forsberg, 2001). Care needs to be taken in administering this type ofheat treatment to avoid damaging seed quality. Klein and Hebbe (1994)

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found that short hot water treatments of tomato seeds produced plants thatwere 20 percent taller 30 days after sowing, but the beneficial results werenot retained after three months of storage at 5°C.

Biopriming

Several researchers have investigated the use of so-called bioprimingtechniques, by including beneficial microorganisms in the priming pro-cesses as a crop delivery mechanism or to control disease proliferation dur-ing priming itself. For example, Warren and Bennett (2000) added Pseudo-monas aureofaciens as a biological control organism in combination withan osmopriming treatment to control Pythium ultimum in tomato seedlings.Matrix priming and hydropriming are also suitable delivery mechanismsfor beneficial microorganisms, akin to solid-state fermentation (see Mc-Quilken, Halmer, and Rhodes, 1998).

Promotive and Retardant Substances

Many studies report the benefits of gibberellins, ethylene, and/or cyto-kinins such as benzyl adenine in combination with priming, e.g., for celery(Brocklehurst, Rankin, and Thomas, 1983) and for the O2-enriched osmo-priming of bedding plant species (Finch-Savage, 1991). Adding such plantgrowth regulators during priming can improve the germination perfor-mance of some seed species or lots compared to either treatment alone.

Alternatively, treatment with growth retardants has been advocated todwarf the growth habit of transplants, such as bedding plants, which tend todevelop an etiolated growth habit, especially if grown in low-light environ-ments. For instance, Souza-Machado and colleagues (1996) reported thatseed priming with a triazole (50 ppm paclobutrazol) in tomato cultivars pro-duced seedlings that were shorter, greener, more uniform, with strongerthicker stems, and higher root:shoot weight ratios than nonprimed controls,though emergence itself was reduced: after five weeks in the field primedseedlings were taller than unprimed controls. Pill and Gunter (2001) foundsimilar dwarfing responses in marigold seeds matrix-primed with paclo-butrazol.

Drying

The technique and rate of drying after priming are also very important tosubsequent seed performance. Slow drying at moderate temperatures is

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generally (e.g., Jum Soon, Young Whan, and Jeoung Lai, 1998) but not al-ways (e.g., Parera and Cantliffe, 1994a) preferable. Various manipulationshave been proposed to extend the storage life of primed seeds. Gurusingheand Bradford (2001) found that a moisture reduction of 10 percent or morewas effective in extending the longevity of hydroprimed tomato seeds. Heatshock is another tactic. For several species Bruggink, Ooms, and van derToorn (1999) have found that greater longevity is obtained by keepingprimed seeds under mild water and/or temperature stress for several hours(e.g., tomato) or days (Impatiens) before drying. These methods are verysimilar to those used to induce desiccation tolerance in just-germinatedseeds, e.g., in cucumber radicles (Leprince et al., 2000).

Electromagnetic Treatments

Advantageous germination and seedling growth responses after treatingseeds with continuous, intermittent, or rapidly pulsed exposure of seeds tostationary or alternating magnetic fields (typically up to about 0.25 to 1.0Tesla) or electric fields (up to 100 kV/m or more) have been known forsome time (Heydecker and Coolbear, 1977). These phenomena continue toreceive research attention, though not prominently in the seed physiologyliterature (e.g., see Kornarzynski and Pietruszewski, 1999; de Souza Torres,Porras Leon, and Casate Fernandez, 1999; Carbonell, Martinez, and Amaya,2000). The mechanisms underlying these intriguing responses, and thereproducibility of possible practical treatments based on them, remain to beinvestigated, and the subject will not be discussed further here.

PHYSIOLOGICAL RESPONSES TO ENHANCEMENT

From the previous section it can be appreciated that priming should beseen as taking the process of germination to various degrees, selecting froma continuum of water potentials, etc., and different durations and dryingprocedures. In practice therefore, priming is not a single treatment, butrather is the result of a choice between options. What constitutes optimalpriming for a given seed lot is a compromise that will vary depending on re-search or commercial circumstances—including how quickly seed is to besown. Because methods and conditions differ greatly, it is hard to generalizeabout responses, and care must be taken in drawing conclusions from thescientific literature about the mechanisms of priming.

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Water Relations and Kinetics of Germination

Water Uptake

Seed germination in the strict sense is commonly defined as those eventsthat begin with water uptake and end with the penetration by the embryonicaxis (usually the radicle) through the structures surrounding the embryo.Seeds with permeable seed coats that are dry at maturity classically displaya triphasic time course of water uptake: seed imbibition (Phase 1), reflect-ing the initial rapid absorption of water by the dry seed; a period of variablelength in which seed water content is relatively constant or only slowly in-creasing (Phase 2), which may be greatly prolonged depending on the de-gree of dormancy; and a resumption of water uptake (Phase 3) associatedwith expansion and growth of the embryo after germination is complete(Bewley and Black, 1994).

Hydrothermal Time Models of Germination and Priming

Germination. Mathematical population models have been successfullydeveloped in the past two decades to unify germination behavior in terms ofwhat seem to be physiologically meaningful water potential ( ) and tem-perature (T) thresholds, and have been extended to cover dormancy, growthregulators, and—for our purposes in this chapter—priming. It might behoped that tests based on a model which describes the key variables for aseed lot (some of which might turn out to be nearly constant at the species orcultivar level) could provide a simple guide to predict the operational pa-rameters for optimal priming, which would be very valuable for the seed in-dustry. These so-called hydrothermal time models will be outlined onlybriefly here since they are covered in depth by Finch-Savage in Chapter 2.In addition, the reader can see Bradford (1995), Cheng and Bradford(1999), and references therein for comprehensive reviews.

Most seed lots exhibit optimum temperatures within the thermal windowin which seeds are capable of full germination, which is defined as the pointat which time taken for germination of a certain fraction g is minimum. Atsupraoptimal temperatures, as T increases germination appears to declinelinearly for each fraction and falls to zero at its upper limit Tc(g), i.e., thetime taken for germination becomes infinite. Similarly, there is a minimumtemperature at which germination falls to zero. The germination timecourse of a seed lot sums the performance of individual seeds, which haveinherently variable properties. According to hydrothermal-time thinking,an individual hydrated seed below its optimum temperature completes ger-

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mination when it has accumulated the heat units characteristic of its rank (g)in the seed lot. (It is assumed that individual seeds in the population wouldgerminate in the same order over the range of conditions covered.) Temper-ature affects germination rates (GRn, the reciprocal of the time it takes forthe nth radicle to emerge) on a thermal time basis: the T in excess of a baseor minimum temperature (Tb) multiplied by the time to a given percent ger-mination (tg) is a thermal-time constant ( T), which differs for each seedfraction. Somewhat analogously, Gummerson (1986) suggested that re-duced water potential delays germination on a hydrotime basis; the in ex-cess of a threshold base water potential ( b, which differs for each seedfraction) multiplied by tg has a constant value for all seed fractions, at a con-stant temperature. He further proposed that germination responses to T and

could be combined into a single expression, using a hydrothermal timeconstant ( HT), defined for all fractions as [(T – Tb)( – b)]· tg. By rear-rangement of this equation, if the constants and variables are known, thegermination rate of a fraction can be predicted: GR = [(T – Tb)( – b)]/( HT). In theory, germination data obtained at just two water potentialswould be sufficient to determine Tb and b(g), but more measurements maybe needed in practice to give greater precision. Although it was developedfor constant conditions, the model has been adapted to describe and predictbehavior in irregularly changing temperature and water potential environ-ments by integrating performance over a number of time intervals—as dis-cussed in Chapter 2.

This hydrothermal time model has been found to match sets of germina-tion time courses of nondormant seeds quite well, e.g., in tomato, lettuce,onion, mungbean, and melon, though more research is needed to cover awider range of species and environmental conditions and to understand thegenetic components (Dahal, Bradford, and Haigh, 1993; and see Welbaum,Bradford, et al., 1998; Cheng and Bradford, 1999). The general situation isas follows: (1) there is relatively little variation in Tb among individualseeds within a seed lot, or even within a species, although further work isneeded; (2) the variations in b between fractions in a seed lot are approxi-mately normally distributed with

b; but (3) agreement can be poor for

some time courses—e.g., behavior can deviate close to Tb and b, and thevalues of each can vary depending upon the environmental or hormonalconditions, including perhaps during test incubations.

In mathematical terms, then, the objectives of seed enhancement couldbe said to be one or more of the following: (1) to raise the optimum tempera-ture and the upper limit Tc(g); (2) to lower b (which determines when andwhether a given seed will germinate under specific environmental condi-tions); (3) to minimize HT, H, and T(50) and the distribution of b(g) and

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T(g), which determine the rate and uniformity of germination. Three ex-amples will illustrate responses that have been observed.

• Osmopriming of tomato cultivars, for instance, increased GR at all T> Tb and > b(g) without lowering Tb or b, (i.e., it reduced T and

H), but it also increased the variance in tg (Dahal, Bradford, andJones, 1990; Dahal and Bradford, 1990). Priming at –0.5 MPa > >–1.0 MPa appeared to shift distributions of b(g) to lower values,i.e., allowing subsequent germination to occur at a that initiallywould have blocked radicle emergence, but there was no shift in

b(g) distributions after priming at > –0.5 MPa (Cheng and Brad-ford, 1999).

• In contrast, osmopriming of a mature muskmelon cultivar decreasedTb and b but left H unaffected (Welbaum and Bradford, 1991). Im-mature seeds were more responsive to priming than mature seeds,suggesting that the overall degree of response would be strongly in-fluenced by the distribution of seed ages within a lot.

• The model has also been applied in studies of germination near theupper temperature limit Tc(g). In lettuce seeds, as temperature wasraised up to this point, germination became more and more sensitiveto reduced water potentials; in hydrotime language, the b(g) distri-bution became progressively more positive. Osmopriming resultedin smaller increases in b(g) near the temperature limit, as well as re-ducing the hydrotime requirement for germination (Bradford andSomasco, 1994).

During priming. The fact that germination can be substantially advancedby priming seeds with water potentials < b (as well as > b) or at tempera-tures close to Tb, led to the proposal (see Bradford and Haigh, 1994) that theconcept of hydropriming time or hydrothermal priming time may be appli-cable—invoking the idea that seeds can also accrue hydrotime and/or heatunits in relation to a different base potential min and temperature Tmin(lower than b and Tb), even though they cannot complete germination (un-less they are being incubated for long enough above b and Tb). The resul-tant median germination rate (GR50) after a priming duration of tp is theoret-ically, GR50 = GRi + k'[(T–Tmin)( – min)] · tp; where k' is the inverse of thehydrothermal priming time constant HTP, and GRi is the initial median ratefor unprimed seeds, determined from the hydrothermal model expression.This model therefore provides a way of comparing a wide range of primingtreatments on a common basis, across temperatures, water potentials, and

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durations. Theoretically, knowing the values of Tb, Tmin, HT, HTP and, es-pecially, the means and distributions of b(g) and min might be practicallyuseful before deciding to prime a seed lot.

In initial tests the hydrothermal priming time model proved capable ofexplaining a large part of the variance in tomato seed primed over a range ofwater potentials (Dahal, Bradford, and Haigh, 1993). However, in a recentin-depth critical study, Cheng and Bradford (1999) concluded that, despiteproviding a useful quantitative description of priming responses, the initialwater relations characteristics of the tomato seed lots they studied did notpredict the responses very precisely. They found that mean values of min =–2.4 MPa and Tmin = 9.1ºC (both assumed for simplification to be the samefor all seed lot fractions) fitted five out of six lots, and that b(g) values laybetween –0.6 and –1.1 MPa and Tb between 12 and 13.5ºC. But all parame-ters varied between seed lots, and in some of them Tmin values were unreli-able, suggesting that other factors remained to be accounted for. Seeds witha faster initial germination rate seemed to have lower min values, meaningthat a shorter duration of priming would be required to achieve a given de-gree of advancement.

It is apparent that, at least in the species and cases studied so far, thesehydrothermal time models give only approximate predictions and for prac-tical purposes do not appear by themselves to have the power to reliablyprescribe how to prime individual seed lots. Refinements, or alternativemodels, may improve the outlook in future. For instance, Rowse, McKee,and Higgs (1999) have addressed one imperfection of the hydrothermalpriming time model—its prediction that increase in resulting germinationrate is proportional to priming time (tp), whereas in reality at low water po-tentials the rate tends to reach a maximum that does not increase with fur-ther increases in duration—and have avoided the complications of theboundary condition at water potentials close to b. They have proposed in-stead a new water relations model of seed germination using a differentvariable (the “virtual osmotic potential”) to integrate the effect of constantor varying water potentials, without having to distinguish between primingand germinating potentials, and with certain assumptions this model seemsto fit the performance of carrot and onion.

Despite their imperfections, the apparent general validity of the hydro-time and hydrothermal priming time models has given further impetus to re-search directed toward relating temperature and, in particular, water poten-tial thresholds to the forces that drive and/or hold back radicle emergencefrom seeds, and to their biological determinants.

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Completion of Germination

Seed Structural Constraints

Seeds (meaning both botanical true seeds and dry fruits) vary consider-ably in their internal morphology, as well as their conspicuous externalform, which dictates how embryos enlarge and emerge from the seed, and isreflected in the physiological and biochemical germination mechanisms. Inhis classic comprehensive descriptive survey, Martin (1946) distinguished12 gross anatomical types within gymnosperm and angiosperm genera,based on structure, organization, and compositional characteristics. Em-bryos may be (1) tiny, small, or dominant compared to the whole seed;(2) narrow, broad or spatulate, straight, curved, coiled, bent, or folded; and(3) located around the periphery, at the end or in the center, more or less sur-rounded by or sandwiched between the endosperm or perisperm tissues,which themselves may be living or wholly or partly dead, and have soft orhard textures.

In many seeds of agricultural importance, embryos are relatively uncon-strained by surrounding tissues, such as in cereals, Brassicas, and many le-gumes and grasses (excepting cases of coat-imposed dormancy), and inthese seeds germination involves only the onset of embryo growth. In manycrop seeds, however, confining structures such as endosperm, testa, andpericarp contribute substantial mechanical barriers to embryo growth. Inmature celery and carrot seeds, for instance, the embryo is underdevelopedand entirely embedded in the endosperm and must grow about two or threetimes at its expense by both cell expansion and cell division before visibleradicle emergence occurs (Karssen et al., 1990; Gray, Steckel, and Hands,1990). This embryo growth pattern, along with the variable presence of en-dogenous inhibitory materials, accounts for the typically slow germinationin umbelliferous species. The endosperm also restrains the expansion ofmany seeds with relatively large embryos and is understood to be a majorphysiological determinant of the hydrotime threshold water potential, e.g.,in tomato and pepper seeds in which the endosperm is a substantial tissue,and in lettuce where it is reduced to a thin but tough envelope layer—situa-tions likened by Welbaum, Bradford, and colleagues (1998) to a rigid outertire confining the pressurized inner tube of the embryo.

At the biochemical and cytological levels, then, germination and prim-ing mechanisms may differ considerably between seed structural types dueto the nature of the embryo and its enclosing tissues. Seed populations natu-rally conceal substantial differences in structure and physiological state,e.g., due to indeterminate flowering and seed developmental patterns, and

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often genetic variation as well, which determine the spread in time from oneseed to another to complete germination.

Embryo Growth and Endosperm Cell-Wall Degradation

Plant cell growth is commonly accepted to be the result of the accumula-tion or generation of solutes within cells and osmotic water uptake, whichgenerates sufficient turgor pressure to drive cell wall extension. The pri-mary cell wall, whose main load-bearing component is a network of cellu-lose microfibrils tethered by hydrogen bonds to xyloglucan chains, is be-lieved to yield in response to turgor by mechanisms mainly involving theactivity of -glucanases, xyloglucan endotransglycosylases (XETs), ex-pansins, and hydroxyl radicals (see Cosgrove, 1999, for access to the largeliterature in this area). However, it is not yet known what determines thestart of cell wall loosening in germinating seeds and the generation of suffi-cient turgor to drive radicle elongation to complete germination, as well ashow these properties are distributed between individual extending cells. Forseeds imbibed in water, direct measurements (e.g., in lettuce, melon, to-mato) have indicated that embryo turgor per se would seldom be limitingfor germination, as embryo osmotic potential values are generally quitenegative ( less than –2.0 MPa) (Welbaum, Bradford, et al., 1998). Eitherthe radicle cell walls are too rigid or the structures surrounding the radicleprevent it from expanding.

One key mechanism regulating the timing of radicle emergence in seedswith enveloping endosperms is thought to be enzymatic weakening of therestraining tissues. Many species studied have endosperm cell walls rich in

-(1 4)mannan polysaccharides (thought to be galacto-mannans or ga-lacto-glucomannans), and much recent research focus has been placed onthe role of endo-ß-mannanase as the most prominent likely candidate forlowering the mechanical restraint by cleaving the polysaccharide backbonechain. Considerable evidence now suggests that -mannanase activity is in-deed involved in radicle emergence in these species, although it is doubtfulthat the enzyme is the sole determinant of the process. (The enzyme is alsoinvolved in mobilizing the endosperm as a food reserve, a process thatmainly occurs after germination is completed.) The subject of enzymaticendosperm cell wall weakening during germination has been reviewed indetail by Black (1996), Bewley (1997), and Welbaum, Bradford, and colleages(1998) and will only be outlined here.

The relationship between endosperm cap weakening, the degree of-mannanase activity and the time to germination after priming has been

most thoroughly studied in tomato, which has become a well-studied exper-

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imental system due to advantageous features such as its size allowing easydissection and the availability of PGR response mutants (see references inToorop, van Aelst, and Hilhorst, 1998; Welbaum, Bradford, et al., 1998). Insummary, there are few cases in tomato where germination is observedwithout at least some -mannanase activity being present in the micropylartip endosperm, but high -mannanase activity does not ensure that germina-tion will occur, or vice versa. Primed and redried seeds develop an internalfree space between the embryo and endosperm, and the most rapid germi-nating seeds have the most extensive free space, observed nondestructivelyusing X-radiography (Downie, Gurusinghe, and Bradford, 1999), and a ger-mination-specific -mannanase gene is expressed in the micropylar endo-sperm cap region (Nonogaki, Gee, and Bradford, 2001). However -man-nanase activity has been found to vary startlingly, by at least 1000-fold,even among individual homozygous inbred seeds (Still and Bradford,1997), and it is necessary to work with individual seeds to get a clear pictureof what is happening. A strong correlation has been found during osmo-priming between the lowering of the mechanical restraint, the increase in

-mannanase activity, and the appearance of ice crystal-induced porosity,which was taken to reflect cell wall hydrolysis (Toorop, van Aelst, andHilhorst, 1998). However, the magnitude of these changes in relation to ger-mination advancement differed between priming conditions, even thoughgermination was advanced by all of them. In endosperm caps measured sin-gly, the restraint decreased and the enzyme activity increased during osmo-priming at –0.4 MPa, but neither property changed during priming at –1.0MPa, and two subpopulations could be distinguished at –0.7 MPa with andwithout changes. (To make matters worse, seed lots of the cultivar used inprevious studies showed different absolute patterns of ß-mannanase activityacross a similar range of water potentials.) The authors concluded that low-ering of the endosperm restraint during priming positively affects the ger-mination rate of primed seeds but is not a prerequisite for rapid germination.The finding that priming tomato seeds at more negative osmotic potentialsdecreases the base water potential b suggests that a different mechanismbecomes more prominent under these priming conditions than at higher po-tentials—the accumulation of solutes in the embryo, possibly, instead of re-quiring a substantial weakening of the force to puncture the endosperm cap.

Apparently contradictory conclusions have been reached in explaininghow priming alleviates thermoinhibition in lettuce. Bradford and Somasco(1994) concluded from the modeling of water relations that the beneficialeffects appeared to occur primarily in the embryo, by lowering the embryoyield threshold sufficiently to compensate for the increased endosperm re-sistance, rather than affecting the surrounding endosperm/pericarp enve-lope tissues. Sung Yu, Cantliffe, and Nagata (1998) found that the endo-

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sperm in thermotolerant cultivars had a lower resistance to puncture thanthermosensitive ones, and furthermore priming reduced the initial forcenecessary to penetrate the seed and endosperm in several genotypes. Theseauthors concluded that, for radicle protrusion to occur, there must first be adecrease in the resistance of the endosperm layer to the turgor pressure ofthe expanding embryo. Circumstantial support comes from the finding thatseeds from thermotolerant lettuce genotypes had higher endo- -mannanaseactivity before radicle protrusion at 35ºC than thermosensitive ones. En-zyme activities increased during priming of thermosensitive varieties andtherefore might be used as an indicator of priming (Nascimento, Cantliffe,and Huber, 2000).

Much remains to be understood about the mechanisms and physiologicalfunction of weakening the layers that surround embryos. Even in mannan-rich seeds, for instance, -mannanase is by no means the only key enzymeor process likely to be involved. In germinating tomato seed, Bradford andcolleagues have already identified polygalacturonase, arabinosidase, and ex-pansin genes that are expressed predominately in the endosperm cap andradicle tip regions, and appear to have roles in cell wall modification or tis-sue weakening (Sitrit et al., 1999; Bradford et al., 2000) along with genesfor -1,3-glucanase and chitinase which are thought to have other functions(Wu et al., 2001). Similarly a -1,3-glucanase appears to be important inrupturing the tobacco endosperm, which is the limiting step in seed germi-nation (Leubner-Metzger and Meins, 2000). Interestingly, an extensin-likegene is specifically expressed during germination at the micropylar end ofthe single-cell-layer endosperm of Arabidopsis, though its function is notyet known (Dubreucq et al., 2000). The expression of some of these late-germination-stage genes may therefore prove of value as markers for prim-ing.

Embryo Cell Division

As already mentioned, seeds such as carrot and celery have rudimentaryimmature embryos, which grow before the radicle emerges by a combina-tion of cell division and cell expansion, and both processes also occur, to alesser degree, during priming at lower water potentials (Karssen et al.,1990). By contrast, in seeds with proportionally large embryos the generalcase seems to be that cells do not complete mitosis, or even grow apprecia-bly, before the embryo structures emerges from the seed. Priming seems tohave no appreciable effect on either cell size or number in these cases, e.g.,in leek and onion (Gray, Steckel, and Hands, 1990).

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In dry tomato and pepper seeds most embryonic nuclei embryo are in thequiescent presynthetic G1 phase, with 2C amounts of DNA. De Castro andcolleagues (2000) have demonstrated by elegant histochemistry that DNAsynthesis during tomato germination in water starts in the radicle meristemtips before cell expansion begins, and the activation pattern then spreadstoward the cotyledons as progressively more nuclei enter the G2 (4C)phase. At the same time, -tubulin protein appears, accumulates, and is as-sembled into the microtubular cytoskeletal networks involved in the mitoticapparatus and establishing the plane of cell division. The situation is similarin sugar beet because in a substantial proportion of seeds a number of theradicle tip cells can enter the G2 phase, depending on environmental condi-tions during seed development, especially after heavy rainfall, and on seedmaturity at time of harvest: on these grounds the G2:G1 ratio has been sug-gested as an indicator of the physiological status of a seed (Sliwinska,2000). Here too, however, nuclear DNA contents increase one to two daysafter the start of imbibition, preceded by the accumulation of -tubulin (Sli-winska et al., 1999), and DNA replication also occurs during priming(Redfearn and Osborne, 1997). Detailed studies of cell-cycle events in to-mato, pepper, and sugar beet using flow cytometry and other techniqueshave revealed that the beneficial effects of priming are associated with theonset of replicative DNA synthetic processes in radicle meristem nuclei,leading to cells stably arrested in the G2 phase after drying. Priming-induced DNA replication and accumulation of -tubulin have thereforebeen suggested as molecular markers for measuring the progression ofevents preceding radicle protrusion (Lanteri et al., 2000).

However, the relationship of cell cycle activity in radicle meristems priorto emergence and the degree of subsequent priming response has proved tobe not at all straightforward. In tomato and pepper, the degree of change inDNA replication was found to vary considerably among similarly osmo-primed seed lots, even of the same cultivar, though all displayed more rapidradicle emergence; in some lots, the frequency of 4C nuclei increased inproportion to the accumulated hydrothermal priming time, while in otherlots no increase was detected following priming (Lanteri et al., 1994;Gurusinghe, Cheng, and Bradford, 1999). In extensive studies on a singletomato seed lot, a positive linear relationship was found between the fre-quency of 4C nuclei (which could be as high as about 30 percent) and theimprovement in median subsequent germination time when priming wasperformed at up to 25°C and above –1.5 MPa; but there was no correlationbetween the two after priming at higher temperatures or at –2.0 MPa, atwhich germination rates were improved without generating any increase in4C signals at all (Özbingöl et al., 1999). Similarly in pepper, a range of os-motic treatments induced different frequencies of radicle tip nuclei to enter

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the synthetic phase despite producing very similar effects on germinationrate (Lanteri et al., 1997). Also there was no consistent relationship betweenthe frequencies and rates in cauliflower seeds after aerated hydration orosmopriming (Powell et al., 2000). In these three species at least, therefore,entry into G2 is not essential for germination advancement, especially in“suboptimal” priming conditions, and 4C:2C ratios are not a general mea-sure of the efficiency of priming. Indeed, considerable increases in germi-nation rates can be observed in the absence of any increases in 4C nuclei.

Lanteri and colleagues (2000) investigated the expression of -tubulin inthe root tips of pepper seeds as a complementary marker for priming. Con-centrations of the protein, which increased and declined during seed devel-opment and were undetectable in mature dry seeds, accumulated prior toDNA replication after osmopriming for different durations at two water po-tentials, apparently by de novo synthesis. Approximately the same amountof -tubulin was observed at the start of nuclear replication in each case, butthere were no clear further increases after that point. This observation ledthe authors to suggest the possibility of using -tubulin expression as an ad-ditional parameter to differentiate the effectiveness of priming treatmentsthat do not induce nuclear replication.

Energy Metabolism

The drying of imbibed seeds may have profound and damaging effectson respiratory metabolism during water removal and after subsequentreimbibition. Using noninvasive photoacoustic techniques, Leprince andcolleagues (2000) showed that dehydration induces imbalanced metabo-lism before membrane integrity is lost in desiccation-sensitive cucumberand pea radicles germinated in water. Compared to desiccation-tolerant or-gans, CO2 production was much increased before and during dehydration;acetaldehyde and ethanol also appeared, and their emissions peaked wellbefore the loss of membrane integrity, but these could be significantly re-duced when dehydration occurred in 50 percent O2 instead of air. Acetal-dehyde was also found to disturb the phase behavior of phospholipid vesi-cles measured by infrared spectroscopy, suggesting that it may aggravatemembrane damage induced by dehydration. Thus, desiccation tolerance ap-pears to be associated with a balance between down-regulation of metabo-lism during drying and O2 availability. Acetaldehyde and ethanol produc-tion therefore might prove to be sensitive hazard signals for the overprimingof seed.

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In practice, though, priming may not often bring seeds to the point wheresuch drastic changes occur. Very few studies have been conducted specifi-cally relating to primed seeds. Corbineau and colleagues (2000) found thatosmopriming raised both the energy charge (EC) and the ATP:ADP (adeno-sine triphosphate:adenosine diphosphate) ratio in tomato seeds, with themaximal effect obtained in osmopriming atmospheres containing morethan 10 percent oxygen; these increases were partially retained after dryingand were associated with much more intense respiratory metabolism duringthe first hours of subsequent imbibition in water.

Early Proteins Reserve Mobilization

A relationship has been found in sugar beet seeds between germinationperformance after priming and the first stage of mobilization of the 11-Sglobulin storage protein, which consists of A and B subunits attached by adisulfide bond. Both hydropriming and osmopriming substantially increasedthe solubilization of the B subunit, still linked to a fragment of the endo-proteolytic cleaved A-chain (Job et al., 1997); this accounted for up to ap-proximately 30 percent of the total B-subunit content in mature seeds,which in turn only was decreased by further proteolysis after radicle emer-gence. Similar behavior has been detected in Arabidopsis, in which degra-dation products of the 12S cruciferin B subunits accumulated during prim-ing (Gallardo et al., 2001). Hydropriming sugar beet also reduced the rangeof soluble B-subunit content among individual seeds from 160-fold in un-treated seeds to only fivefold (Bourgne, Job, and Job, 2000).

Job, Kersulec, and Job (1997) have therefore proposed that B-subunitsolubilization can be used as a protein marker for the optimization of prim-ing in sugar beet. Evidence supporting the robustness of this potentialmarker has come from the observation that, in two types of priming, therange of temperatures and oxygen concentrations that were effective inspeeding germination were very similar to those which solubilized B sub-units (Capron et al., 2000). A complication arises because soluble B subunitshave been detected in “late mature” harvested seeds in some lots, suggest-ing again that events associated with germination can occur in some cir-cumstances during the last stages of seed development, as already noted inthe previous section for G2:G1 ratios (Sliwinska et al., 1999). Job and col-leagues (2000) have also proposed that the disappearance of biotinylatedprotein can be used as marker of overextended osmopriming, which led to asubstantial drop in germination under the conditions used.

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Desiccation Tolerance and Storability

As a seed lot becomes overprimed, due to too long an exposure at thechosen and T, more and more individuals typically display damaged pri-mary radicle meristems and, although they may complete germination inthe strict sense that the radicles emerge, seedlings may not develop properly(e.g., more abnormal types are detected in the statutory germination test)and so may develop plants with a weakened root system or a damaged shoottip or die before they emerge from the soil. Even though only a small per-cent of seeds in a lot may be affected, such losses are unacceptable for high-value seed in commercial practice. It is therefore valuable to determine safelimits for priming to minimize or preferably avoid these handicaps.

Desiccation tolerance in seeds is a fertile research area, with much focuson processes at the termination of seed development, including the physiol-ogy of recalcitrant seeds which cannot survive drying after development onthe mother plant, recently reviewed by and Pammenter and Berjak (1999)and in Chapters 9 and 10 of this book. Vertucci and Farrant (1995) suggestthat seeds might suffer different types of injury when water is withdrawn:mechanical damage, due to the reduction in cell volume; metabolic dam-age, due to failures in the coordinated regulation at intermediate water con-tents and the failure of protective antioxidant systems; and subcellularstructural damage, due to removal of water intimately associated with thesurface of macromolecular structures such as membranes. It is believed thatdesiccation-tolerant seed tissues require the interplay of a multifactorialsuite of protective mechanisms to prevent these forms of damage and/orpermit their repair on rehydration. Major roles are thought to be played bythe composition of the cytoplasm (including soluble sugars), the presenceof putatively protective molecules including late embryogenesis abundant(LEA) proteins, the efficient operation of antioxidant systems to protectagainst free radicals produced during dehydration that would otherwiselead to oxidative damage to membranes, and the presence and operation ofrepair mechanisms during rehydration (e.g., see Leprince, Hendry, andMcKersie, 1993). The absence or ineffective expression of one or more ofthese mechanisms could determine desiccation sensitivity in primed seedsand could therefore serve as markers for safe priming.

Cell Stabilization: Sugars and LEA Proteins

It has been known for some years that sucrose and oligosaccharides, usu-ally of the galactosyl-sucrose family (raffinose, etc.), occur in relativelylarge amounts in seeds and that their concentrations appear to correlate with

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the longevity of seeds and with the acquisition and loss of desiccation toler-ance (Bernal-Lugo and Leopold, 1995; Koster and Leopold, 1988, Corbin-eau et al., 2000). During germination and priming the content and compositionof intracellular soluble carbohydrates changes; for example, the oligosac-charide:sucrose ratio is reduced in primed pea and cauliflower (Hoekstraet al., 1994; Buitink, Hoekstra, and Hemminga, 1999). Soluble sugars aretherefore widely understood to be important components in stabilizing cel-lular integrity during dehydration—though the mechanisms by which theydo so are still a matter for research and debate—and are thus prominent can-didates to consider as a contributing factor to the performance of primedseed after drying.

Cytoplasm. In air-dry storage conditions the cytoplasm of seeds andother anhydrobiotic organisms enter an aqueous glassy state; i.e., it be-comes a solidlike liquid well below its normal freezing (crystallizing) point.The extremely high viscosity and low molecular mobility of the cytoplasmunder these conditions are believed to be major factors in imparting longev-ity to anhydrobiotes, by restricting the rate of detrimental reactions associ-ated with aging and stabilizing macromolecules, such as membranes, pro-teins, and DNA. It is thought, for instance, that direct measurements ofmolecular mobility might eventually lead to realistic predictions of longev-ity of seeds, such as those stored at low temperatures (Buitink, Hoekstra,and Hemminga, 2000). At one time it was suspected that supersaturated so-lutions of soluble sugars were the primary elements responsible for theglassy state in seeds, but recent physicochemical studies have revealed thatthe situation is more complex, and glycosides, larger carbohydrates (e.g.,maltodextrin), and proteins also contribute to the property (Leopold, Sun,and Bernal-Lugo, 1994).

Membranes. Removal of water from membrane surfaces in desiccation-sensitive plant cells at low water contents causes the liquid crystalline lipidbilayer to convert into gel phase domains and, although they are readily re-versed on rehydration, these transformations are associated with symptomsof injury and lethal effects, including extensive solute leakage and reorgani-zation of the membrane protein complex. In desiccation-tolerant tissues, incontrast, according to the water replacement hypothesis (Crowe, Hoekstra,and Crowe, 1992), sugars and sugar alcohols replace the structural waterthat is normally hydrogen bonded to the membrane surfaces and macro-molecules, and thereby maintain the correct of polar head group spacing ofthe membrane lipid and provide the hydrophilic interactions necessary tomaintain the liquid-crystalline phase. However, a recent review (Hoekstraet al., 1997) has concluded that sugars may not be particularly effective invivo in this way and proposes instead that the bilayer is stabilized by the mi-gration of amphipathic molecules (e.g., flavinols) into membranes as water

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is lost, which substantially lowers the water content at which membranelipids undergo the liquid-to-gel phase change. Bryant, Koster, and Wolfe(2001) have argued that the presence in the cytoplasm of small solutes thatcan form glasses, such as sugars, could limit the close approach of mem-branes and thereby diminish the physical stresses that could otherwisecause lipid fluid-to-gel phase transitions to occur during dehydration.

Whatever the stabilization mechanisms and the role of sugars in them,changed macroscopic physical properties of intracellular glasses do notseem to offer an explanation by themselves for the reduced longevity ofprimed seed. Differential scanning calorimetry revealed no changes in glasstransition temperature (i.e., when the matrix “melts”) associated with theosmopriming of pea, Impatiens, and pepper seeds. Nor was there any signif-icant difference in molecular mobility, as determined by electron paramag-netic resonance spectroscopy of a spin probe introduced into the cytoplasm(Buitink, Hoekstra, and Hemminga, 1999; Buitink, Hemminga, and Hoek-stra, 2000). Nevertheless, a circumstantial but perhaps relevant parallel canbe drawn with the behavior of abscisic acid-pretreated carrot somatic em-bryos, which survive slow dehydration much better than fast, as primedseeds tend to do. Using in situ infrared microspectroscopy, Wolkers andcolleagues (1999) found that fast drying resulted in much weaker averagestrength of hydrogen bonding at room temperature, a less clearly definedglassy matrix, apparently “less tight” molecular packing, and greater extentof protein denaturation than slow drying. LEA transcripts were also ex-pressed after slow drying, suggesting a role in conferring stability withinthe glassy matrix.

Sugar composition is not consistently related to storage life performanceof primed seeds. Gurusinghe and Bradford (2001) found that the shortpostpriming heat treatments that substantially restored longevity to hydro-primed tomato seeds only slightly changed sucrose and oligosaccharide(planteose) content. The suggestion here instead was that heat-shock pro-teins might be involved in the response, supported by the observation thatthe effectiveness of the heat treatment was correlated with expression of aconstitutively expressed lumenal stress protein (BiP), a highly conservedmember of the hsp 70 family associated with the endoplasmic reticulum(Gurusinghe, Powell, and Bradford, 2002).

Free Radicals and Oxidative Damage

It is widely recognized that toxic active oxygen species (AOS), e.g.,superoxide radicals and H2O2, are produced as products of mitochondrialrespiration and glyoxysomal lipid degradation, and are removed within

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plant cells by antioxidative enzymes, e.g., superoxide dismutase (SOD) andcatalase (CAT), etc., which scavenge free radicals before they disruptbiomolecules.

One major harmful effect of free radical reactions is believed to be theaccumulation of free fatty acids and other lipid-degradation products inmembrane bilayers, which increase the lipid phase transition temperatureand cause the irreversible formation of gel phase domains, which are lethalwhen the cell is rehydrated (McKersie, 1991). Bailly and colleagues (2000)found in sunflower seeds that malondialdehyde (MDA) content—a mea-sure of the degree of lipid degradation—remained unchanged during osmo-priming (–2.0 MPa), while activities of SOD and CAT increased strongly.Furthermore, although MDA concentrations increased markedly after dry-ing, they declined again during six hours from the start of reimbibition,compared to an increase in control imbibed unprimed seed. This supportsthe idea that the enzymatic antioxidant defense system operates efficientlyin sunflower seeds to scavenge AOS produced during osmopriming and thatthe system survives in an enhanced state in dried primed seeds to operateduring the first hours after subsequent reimbibition, though it cannot copewith the effects of the intervening dehydration stage itself. The authors alsosuggest that the CAT isoenzyme pattern may be a good marker of whethersunflower seeds have been primed under their conditions.

Another possibility that might be investigated in primed seeds is whetherreducing sugars, such as glucose and fructose, which decrease in embryosas seed matures and acquire desiccation tolerance, and increase followinggermination, will cause a reaction with metal ions such as Fe to generate ox-idizing agents through the Maillard and Amadori rearrangement reactionsof carbonyl groups with free amino groups in proteins, which can causenonenzymatic modification during seed aging (Murthy and Sun, 2000).

DNA Damage and Repair

The ability to repair DNA damage after seeds are rehydrated so that atranscriptionally competent genome is assured has been proposed as an es-sential element of the suite of mechanisms contributing to survival of dehy-drated orthodox seeds (Boubriak et al., 1997). Repair of lesions of the ge-nome that occurs during drying and storage has been shown to be among theearliest events occurring when dry, orthodox seeds are rehydrated, andchanges during priming might impair this ability. van Pijlen and colleagues(1996) speculated that the adverse storage performance of an osmoprimed(compared to a humidified or hydroprimed) tomato seed treatment could beexplained by the fact that more nuclei in the osmoprimed embryonic root

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tips had replicated their DNA, and Lanteri and colleagues (1997) andPowell and colleagues (2000) have made similar observations in osmo-primed pepper and hydroprimed cauliflower. Perhaps the detrimental ef-fects of overpriming are associated with a decreased ability to repair DNAas cells enter the S phase of the cell cycle and progress toward the G2 phaseafter subsequent rehydration.

In another hypothesis that remains to be tested, Boubriak and colleagues(2000) have pointed out the danger that, under certain low water potentialregimes during the priming or subsequent drying of imbibed seeds, DNAmight be subjected to irreparable enzymatic cleavage to nucleosome oligo-mers, as can occur during the accelerated aging of rye grains.

Conversely, during the process of the osmopriming itself, DNA and itssynthesizing systems can be repaired and damaged rRNA replaced (Bray,1995). McDonald, in Chapter 9, discusses the ability of priming to over-come such low vigor effects that result from seed deterioration in storage.

ECOLOGICAL ASPECTS OF SEED HYDRATION

Seed banks in the soil naturally experience a variable environment oftemperature, water potential, oxygen, and other factors, both daily and sea-sonally. Seed burial of numerous species induces physiological changesthat can improve the chances of establishment, allowing the seeds to re-spond to conditions favorable for germination and growth, through the de-velopment or breaking of specific types of dormancy, such as by alternatesoil wetting and drying (Allen and Meyer, 1998; Baskin and Baskin, 1998).However, when a seed population is emerging from dormancy, those indi-viduals that can germinate will have, in hydrotime language, so high a bthat their progress will be slow and unlikely to be completed during a shortwindow of opportunity. Instead they will accumulate hydrothermal timeand survive to germinate more rapidly at the next chance (Bradford, 1995).

The ecological significance of priming existing in nature to increase thechances of successful seedling establishment from the soil seed bank of dif-ferent plant communities remains to be studied, but González-Zertuche andcolleagues (2001) recently obtained circumstantial evidence that seeds ofWigandia urens, a Mexican shrub, do undergo changes while buried in theirnatural habitat that are similar to those seen after osmotic priming in thelaboratory. Both treatments induced physiological changes that were pri-marily expressed in the heterogeneous burial environment, including thesynthesis of heat-soluble proteins, of similar molecular size to LEA pro-teins. It is entirely plausible that the ability of seeds to survive interruptedgermination and be primed is an expression of processes that have evolved

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in the soil seed bank to prepare some species for a rapid, uniform, and suc-cessful colonization of their environments.

CONCLUSIONS AND FUTURE DIRECTIONS

As seed becomes increasingly valuable, by the addition of input and out-put traits through genetic engineering and other breeding techniques, anddue to increasing economic pressures for efficient and environmentallyfriendly crop production systems, the incentive to protect and ensure germi-nation will increase. One trend already underway is that techniques ofpelleting, coating, and priming are being considered commercially forlarge-volume crops, for which they were not previously cost-effective, butwhich demand new larger-scale process engineering approaches.

At the same time the need to understand and improve seed physiologicalquality continues to grow. Though the 1990s were a period of considerableadvance in our understanding of the processes that transform germinationinto the start of growth and the loss of desiccation tolerance, our knowledgeis still based on relatively few species—most extensively, tomato. As far ashydration treatments are concerned, it seems there may be no one explana-tion and no simple universal indicators of the processes operating, partlysince in many respects germination patterns are species specific, as Bray(1995) observed. Bearing in mind too that priming of a given species can beconducted using a range of hydration and drying procedures and a spectrumof water potentials and durations, it is perhaps not surprising that metabolicand cellular events have been found to differ. Also, as Welbaum, Bradford,and colleagues (1998) pointed out, it has been valuable to recognize theproblems posed by pooling large numbers of seeds for biochemical assays,which can obscure important seed-to-seed physiological variation, as noted,for example, by Still and Bradford (1997) and Bourgne, Job, and Job(2000). From the practical seed industry point of view, this understandingcan provide experimental tools to help in the development of new hydrationand drying procedures. What has been perhaps more disappointing—andsetting aside its academic interest—in a production situation in which deci-sions have to be made reliably and often rapidly is the picture that oftenemerges of differences between seed lots in the magnitude or even the exis-tence of changes during treatment (e.g., in cell cycle events or the appear-ance of cell wall degrading enzymes), at least in the much-studied tomatoand pepper. As far as markers for priming and desiccation tolerance are con-cerned, this tends to reduce the hope that there might be tests with the preci-sion required to be in a position to decide between alternative priming ordrying conditions or procedures before treatment starts. There is a better

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prospect of tests which give qualitative indications that a particular dry seedlot has already been primed, but so far these tests by themselves probablycannot indicate the quantitative degree of priming without being combinedwith direct measurement of seed performance in comparison by some formof germination test.

Looking to the future, molecular tools are dramatically enhancing ourknowledge of the biochemical and regulatory pathways underlying thecomplex physiological and developmental process of germination. Geno-mic and transgenic approaches can now establish the timing and identity ofspecifically activated genes and their tissue expression patterns, or the con-sequences of specific gene inactivation, and provide insights into their func-tions. The first fruits of this work are beginning to be seen in seed science,such as the identification in germinating tomato seeds of genes associatedwith cell wall weakening enzymes (mentioned earlier) and connected to theinitiation of embryo growth and stress adaptation (see Bradford et al.,2000). Gallardo and colleagues (2001) conducted a broad proteomic analy-sis of changes occurring during germination and after drying of the modelspecies, Arabidopsis thaliana, whose complete genome is now known.Using protein analysis in combination with sequence databases, these au-thors have identified, among many other changes, a total of eight uniqueproteins that accumulated during a hydropriming and/or an osmoprimingtreatment. One of the many stimulating outcomes from using the potentialwealth of this type of information in classical physiological approachescould be the identification of diagnostic markers, which might be routinelyemployed to interrogate gene expression, e.g., using DNA array or ELISAtechnology, to characterize seed quality and to develop and perhaps opti-mize priming enhancement procedures.

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Lanteri, S., Belletti, P., Marzach, C., Nada, E., Quagliotti, L., and Bino, R.J. (1997).Priming-induced nuclear replication activity in pepper (Capsicum annuum L.)seeds: Effect on germination and storability. In Ellis, R.H., Black, M., Murdoch,A.J., and Hong, T.D. (Eds.), Basic and Applied Aspects of Seed Biology: Pro-ceedings of the Fifth International Workshop on Seeds, Reading (pp. 451-459).Dordrecht: the Netherlands, Kluwer Academic Publishers.

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Martin, A.C. (1946). The comparative internal morphology of seeds. The AmericanMidland Naturalist 36: 513-660.

Massawe, F.J., Collinson, S.T., Roberts, J.A., and Azam Ali, S.N. (1999). Effect ofpre-sowing hydration on germination, emergence and early seedling growth ofbambara groundnut (Vigna subterranea L. Verdc). Seed Science and Technol-ogy 27: 893-905.

Maude, R.B. (1996). Seedborne Diseases and Their Control: Principles and Prac-tices. Wallingford, UK: CABI Publishing.

Maude, R.B., Drew, R.L.K., Gray, D., Petch, G.M., Bujalski, W., and Nienow,A.W. (1992). Strategies for control of seedborne Alternaria dauci (leaf blight) ofcarrots in priming and process engineering system. Plant Pathology 41: 204-214.

McDonald, M. (2000). Seed priming. In Black, M. and Bewley, J.D. (Eds.), SeedTechnology and Its Biological Basis (pp 287-325). Sheffield, UK: Sheffield Ac-ademic Press.

McKersie, B.D. (1991). The role of oxygen free radicals in mediating freezing anddesiccation stress in plants. In Pell, E. and Steffer, K. (Eds.), Active Oxygen/Oxi-dative Stress and Plant Metabolism (pp. 107-118). Rockville, MD: AmericanSociety of Plant Physiologists.

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McQuilken, M.P., Halmer, P., and Rhodes, D.J. (1998). Application of microorgan-isms to seeds. In Burges, H.D. (Ed.), Formulation of Microbial Biopesticides,Beneficial Microorganisms and Nematodes (pp. 255-285). Dordrecht, the Neth-erlands: Kluwer Academic Publishers.

Meikle, R.A.R. and Smith, D. (2000). Seed germination system. U.S. Patent No.6070358.

Murthy, U.M.N. and Sun, W.Q. (2000). Protein modification by Amadori andMaillard reactions during seed storage: Roles of sugar hydrolysis and lipidperoxidation. Journal of Experimental Botany 51: 1221-1228.

Nascimento, W.M., Cantliffe, D.J., and Huber, D.J. (2000). Endo-beta-mannanaseactivity during lettuce seed germination at high temperature conditions. ActaHorticulturae 517: 107-112.

Nascimento, W.M. and West, S.H. (1998). Microorganism growth during musk-melon seed priming. Seed Science and Technology 26: 531-534.

Ni, B.-R. (2001). Alleviation of seed imbibitional chilling injury using polymer filmcoating. In Biddle, A. (Ed.), Seed Treatment: Challenges and Opportunities,Number 76 (pp. 73–80). Farnham, UK: British Crop Protection Council.

Nonogaki, H., Gee, O.H., and Bradford, K.J. (2001). A germination-specific endo--mannanase gene is expressed in the micropylar endosperm cap of tomato

seeds. Plant Physiology 123: 1235-1246.Ollerenshaw, J.H. (1988). Calcium peroxide as a seed coating to alleviate stresses

on crop plants. In Martin, T.J. (Ed.), Application to Seeds and Soil (pp. 285-292).British Crop Protection Council Monograph No. 39. Farnham, UK: British CropProtection Council.

Özbingöl, N., Corbineau, F., Groot, S.P.C., Bino, R.J., and Côme, D. (1999). Acti-vation of the cell cycle in tomato (Lycopersicon esculentum Mill.) seeds duringosmoconditioning as related to temperature and oxygen. Annals of Botany 84:245-251.

Pammenter, N.W. and Berjak, P. (1999). A review of recalcitrant seed physiology inrelation to desiccation-tolerance mechanisms. Seed Science Research 9: 13-37.

Parera, C.A. and Cantliffe, D.J. (1994a). Dehydration rate after solid matrix primingalters seed performance of shrunken-2 corn. Journal of the American Society forHorticultural Science 119: 629-635.

Parera, C.A. and Cantliffe, D.J. (1994b). Presowing seed priming. Horticultural Re-views 16: 109-141.

Petch, G.M., Maude, R.B., Bujalski, W., and Nienow, A.W. (1991). The effects ofre-use of polyethylene glycol priming osmotica upon the development of micro-bial populations and germination of leeks and carrots. Annals of Applied Biology119: 365-372.

Pill, W.G. (1991). Advances in fluid drilling. HortTechnology 1: 59-65.Pill, W.G. and Gunter, J.A., Jr. (2001). Emergence and shoot growth of cosmos and

marigold from paclobutrazol-treated seed. Journal of Environmental Horticul-ture 19: 11-14.

Powell, A.A., Yule, L.J., Jing, H.-C., Groot, S.P.C., Bino, R.J., and Pritchard, H.W.(2000). The influence of aerated hydration seed treatment on seed longevity as

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assessed by the viability equations. Journal of Experimental Botany 51: 2031-2043.

Redfearn, M. and Osborne, D.J. (1997). Effects of advancement on nucleic acids insugarbeet. Seed Science Research 7: 261-267.

Rowse, H.R. (1996). Drum-priming—A non-osmotic method of priming seeds.Seed Science and Technology 24: 281-294.

Rowse H.R. and McKee, J.M.T. (1999). Seed priming. World Patent No. 9608132.Rowse, H.R., McKee, J.M.T., and Higgs, E.C. (1999). A model of the effects of wa-

ter stress on seed advancement and germination. New Phytologist 143: 273-279.Schmolka, I.R. (1988). Seed protective coating. U.S. Patent No. 4735015.Simak, M. (1984). Method for sorting of seed. U.S. Patent No. 4467560.Sitrit, Y., Hadfield, K.A., Bennett, A.B., Bradford, K.J., and Downie, A.B. (1999).

Expression of a polygalacturonase associated with tomato seed germination.Plant Physiology 121: 419-428.

Sliwinska, E. (2000). Analysis of the cell cycle in sugarbeet seed during develop-ment, maturation and germination. In Black, M., Bradford, K.J., and Vazquez-Ramos, J. (Eds.), Seed Biology: Advances and Applications. Proceedings of theSixth International Workshop on Seeds, Merida, Mexico, 1999 (pp. 133-139).Wallingford, UK: CABI Publishing.

Sliwinska, E., Jing, H.-C., Job, C., Job, D., Bergervoet, J.H.W., Bino, R.J., andGroot, S.P.C. (1999). Effect of harvest time and soaking treatment on cell cycleactivity in sugarbeet seeds. Seed Science Research 9: 91-99.

Sluis, S.J. (1987). Process for bringing pregerminated seed in a sowable and forsome time storable form, as well as pilled pregerminated seeds. U.S. Patent No.4658539.

Souza-Machado, V., Ali, A., Pitblado, R., and May, P. (1996). Enhancement of to-mato seedling quality involving triazole seed priming and seedling nutrient load-ing. In Proceedings of the First International Conference on the ProcessingTomato (Recife, Brazil, November 1996) (pp. 71-72). Alexandria, VA: ASHSPress.

Stewart, R.F. (1992). Temperature sensitive seed germination control. U.S. PatentNo. 5129180

Still, D.W. and Bradford, K.J. (1997). Endo- -mannanase activity from individualtomato endosperm caps and radicle tips in relation to germination rates. PlantPhysiology 113: 21-29.

Sung Y., Cantliffe, D.J., and Nagata, R. (1998). Using a puncture test to identify therole of seed coverings on thermotolerant lettuce seed germination. Journal of theAmerican Society for Horticultural Science 123: 1102-1110.

Taylor, A.G., Allen, P.S., Bennett, M.A., Bradford, K.J., Burris, J.S., and Misra,M.K. (1998). Seed enhancements. Seed Science Research 8: 245-256.

Taylor, A.G., Klein, D.E., and Whitlow, T.H. (1988). SMP: Solid matrix priming ofseeds. Scientia Horticulturae 37: 1-11.

Taylor, A.G., McCarthy, A.M., and Chirco, E.M. (1982). Density separation ofseeds with hexane and chloroform. Journal of Seed Technology 7: 78-83.

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Taylor, A.G., Prusinski, J., Hill, H.J., and Dickson, M.D. (1992). Influence of seedhydration on seedling performance. HortTechnology 2: 336-344.

Thornton, J.M. and Powell, A.A. (1992). Short-term aerated hydration for the im-provement of seed quality in Brassica oleracea L. Seed Science Research 2:41-49.

Toorop, P.E., van Aelst, A.C., and Hilhorst, H.W M. (1998). Endosperm cap weak-ening and endo- -mannanase activity during priming of tomato (Lycopersiconesculentum cv. Moneymaker) seeds are initiated upon crossing a threshold waterpotential. Seed Science Research 8: 483-491.

Tsujimoto, T., Sato, H., and Matsushita, S. (1999). Hydration of seeds with partiallyhydrated super absorbent polymer particles. U.S. Patent No. 5930949.

van Pijlen, J.G., Groot, S.P.C., Kraak, H.L., Bergervoet, J.H.W., and Bino, R.J.(1996). Effects of pre-storage hydration treatments on germination performance,moisture content, DNA synthesis and controlled deterioration tolerance of to-mato (Lycopersicon esculentum Mill.) seeds. Seed Science Research 6: 57-63.

Vertucci, C.W. and Farrant, J.M. (1995). Acquisition and loss of desiccation toler-ance. In Kigel, J. and Galili, G. (Eds.), Seed Development and Germination(pp. 237–271). New York: Marcel Dekker.

Warren, J.E. and Bennett, M.A. (1997). Seed hydration using the drum priming sys-tem. HortScience 32: 1220-1221.

Warren, J.E. and Bennett, M.A. (2000). Bio-osmopriming tomato (Lycopersiconesculentum Mill.) seeds for improved stand establishment. In Black, M., Brad-ford, K.J., and Vazquez-Ramos, J. (Eds.), Seed Biology: Advances and Applica-tions. Proceedings of the Sixth International Workshop on Seeds, Merida,Mexico, 1999 (pp. 477-487). Wallingford, UK: CABI Publishing.

Watts, H. and Schreiber, K. (1974). Manufacture of dormant pellet seed by coatingwith non-elastomeric polymer. U.S. Patent No. 3803761.

Welbaum, G.E. and Bradford, K.J. (1991). Water relations of seed development andgermination in muskmelon (Cucumis melo L.): VI. Influence of priming on ger-mination responses to temperature and water potential during seed development.Journal of Experimental Botany 42: 393-399.

Welbaum, G.E., Bradford, K.J., Yim, K.O., Booth, D.T., and Oluoch, M.O. (1998).Biophysical, physiological and biochemical processes regulating seed germina-tion. Seed Science Research 8: 161-172.

Welbaum, G.E., Shen, Z., Oluoch, M.O., and Jett, L.W. (1998). The evolution andeffects of priming vegetable seeds. Seed Technology 20: 209-235.

Wolkers, W.F., Tetteroo, F.A.A., Alberda, M., and Hoekstra, F.A. (1999). Changedproperties of the cytoplasmic matrix associated with desiccation tolerance ofdried carrot somatic embryos: An in situ Fourier transform infrared spectro-scopic study. Plant Physiology 120: 153-163.

Wu, C.T., Leubner-Metzger, G., Meins, F., Jr., and Bradford, K.J. (2001). Class I-1,3-glucanase and chitinase are expressed specifically in the micropylar endo-

sperm of tomato seeds prior to radicle emergence. Plant Physiology 126: 1299-1313.

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SECTION II:DORMANCY AND THE BEHAVIOR

OF CROP AND WEED SEEDS

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Chapter 5

Inception, Maintenance, and Termination of Dormancy in Grain CropsInception, Maintenance, and Terminationof Dormancy in Grain Crops: Physiology,

Genetics, and Environmental Control

Roberto L. Benech-Arnold

INTRODUCTION

Dormancy is the failure to germinate because of some internal block thatprevents the completion of the germination process (Black, Butler, andHughes, 1987). For completeness it should added that dormant seeds can-not germinate in the same conditions (e.g., water, air, temperature) underwhich nondormant seeds do so. Although the adaptive significance of dor-mancy is quite evident for plants living in the “wild” (see Chapter 8), it hasalways been a complication in seeds from plants that are grown as crops. In-deed, a persistent dormancy would prevent the utilization of a seed lot eitherfor the generation of a new crop or for industrial purposes (i.e., malting). Onthe other hand, most crops that originally must have had dormancy havebeen selected so heavily against dormancy throughout their domesticationprocess that seeds are germinable even prior to crop harvest; this frequentlyleads to preharvest sprouting, a phenomenon whose consequences arewidely described in Chapter 6.

Due to the paucity of our knowledge on the genetic, physiological, andenvironmental control of dormancy, it is very difficult to adjust the timingof dormancy loss to a precise and narrow time window (i.e., neither as earlyas to expose the crop to the risk of preharvest sprouting, nor as late as tohave a dormant seed lot at the time of the next sowing or industrial utiliza-tion). Among the cereals, malting barley is possibly the most problematiccrop. The malting process itself requires grain germination, so a low dor-mancy level at harvest is a desirable characteristic because the grain can bemalted immediately after crop harvest, thus avoiding costs and deteriora-tion resulting from grain storage until dormancy is terminated. Therefore,breeders are compelled to work within a narrow margin. In this case, the

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possibility of solving the conflict between obtaining genotypes with lowdormancy at harvest, but not with such an anticipated termination of dor-mancy that leads to sprouting risks, requires a thorough knowledge of themechanisms determining dormancy release in the maturing grain. More-over, it is essential to understand how those mechanisms are genetically andenvironmentally controlled.

Problems derived from either a short or a persistent dormancy are lessfrequent in oil crops, though they do exist. Sprouting, for example, has notbeen reported to occur in the most important oil crops (i.e., soybean, sun-flower, canola); this is in spite of the fact that both soybean and canola seedsare germinable as soon as the grain has undergone desiccation in the motherplant. Soybean and canola seeds develop within legumes and siliques, re-spectively, which must impede direct contact between the grain and rainwater in the field. Sunflower, however, does not sprout because its seeds arehighly dormant at harvest time, and this deep dormancy may persist for sev-eral months. Indeed, having a dormant lot by the time sunflower seeds aresold for sowing is a significant problem that most seed companies face fre-quently.

The aim of this chapter is to discuss the physiology, genetics, and envi-ronmental control of dormancy inception, maintenance, and loss in somegrain crops, namely, cereals and sunflower. It is intended, also, to analyzethe perspectives of controlling the timing of occurrence of these processesthrough manipulation of the genes that regulate the physiological mecha-nisms involved.

PHYSIOLOGY OF DORMANCY IN THE CEREAL GRAIN

Where Is Dormancy Located in Cereal Grains?

Dormancy inception occurs very early in cereals. Embryos are usuallyfully germinable from early stages of development (i.e., 15 to 20 days afterpollination [DAP]) if isolated from the entire grain and incubated in water(Walker-Simmons, 1987; Benech-Arnold, Fenner, and Edwards, 1991;Benech-Arnold et al., 1999). The entire grain, however, reaches full capac-ity of germination well after it has been acquired by the embryo. This coat(endosperm plus pericarp)-imposed dormancy is the barrier preventing un-timely germination, and its duration depends on the genotype and on the en-vironment experienced during maturation and beyond. In summary, thoughcases of embryo dormancy have been reported for grains of some cerealcrops (Norstog and Klein, 1972; Black, Butler, and Hughes, 1987), the du-ration of coat-imposed dormancy determines the timing of acquisition of

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grain germinability. For example, sprouting-susceptible cultivars are thosewhose coat-imposed dormancy is terminated well before harvest maturity.

In some cereals (i.e., barley, rice), glumellae (the hull) adhering to thecaryopsis represents another constraint for embryo germination in additionto that already imposed by endosperm plus pericarp (Corbineau and Come,1980). Benech-Arnold and colleagues (1999) followed the dynamics of therelease from dormancy imposed by the different structures surrounding theembryo in grains from cultivars with short (cv. B1215) and longer-lastingdormancy (cv. Quilmes Palomar). As expected, embryos from both cul-tivars germinated precociously from early stages of development if excisedfrom the entire grain (Figure 5.1a). In both cultivars dormancy imposed byendosperm plus pericarp was steadily overcome at a similar rate throughoutdevelopment (Figure 5.1b). However, although caryopses presented lowdormancy from well before physiological maturity (PM, defined as the mo-ment when the grain has attained maximum dry weight), the presence of thehull prevented grain germination prior to that stage. Hull-imposed dor-mancy started to be removed from PM onward, with a rate that was differentdepending on the cultivar: in ‘B1215’ grains this restriction was removedabruptly, while in ‘Q. Palomar’ grains, the removal occurred at a lower rate(Figure 5.1c).

Hormonal Regulation of Dormancy in Cereal Grains

The Role of Abscisic Acid

Research on the mechanisms of dormancy in the developing seeds ofmany species suggests a strong involvement of the phytohormone abscisicacid (ABA) (King, 1982; Fong, Smith, and Koehler, 1983; Karssen et al.,1983; Walker-Simmons, 1987; Black, 1991; Benech-Arnold, Fenner, andEdwards, 1991; Benech-Arnold et al., 1995; Steinbach et al., 1995; Stein-bach, Benech-Arnold, and Sánchez, 1997). ABA-deficient or -insensitivemutants of Arabidopsis and maize precociously germinate (Robichaud,Wong, and Sussex, 1980; Karssen et al., 1983), and application of the ABA-synthesis inhibitor fluridone has been shown to anticipate the release fromdormancy in developing seeds of some species (Fong, Smith, and Koehler,1983; Xu, Coulter, and Bewley, 1991; Steinbach, Benech-Arnold, andSánchez, 1997). In cereals, the imposition of dormancy to the embryo bythe structures that surround it might be mediated by the high levels of en-dogenous ABA existing in the embryos during grain development (Walker-Simmons, 1987; Steinbach et al., 1995). ABA content in embryos is usuallylow until 15 DAP (Walker-Simmons, 1987; Steinbach et al., 1995; Benech-

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FIGURE 5.1. Germination indexes of (a) embryos, (b) dehulled caryopses, and(c) grains from a sprouting-susceptible (‘B1215’, squares) and a sprouting-resis-tant (‘Quilmes Palomar’, circles) cultivar, harvested at different days after pollina-tion and incubated at 20°C for 12 days. PM and HM are the moments the cropsreached physiological and harvest maturity, respectively. (Source: Adapted fromfigures in Benech-Arnold et al., 1999.)

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Arnold et al., 1999). From that moment onward, ABA content goes up coin-ciding with the acquisition of the capacity of the embryo to germinate if iso-lated from the rest of the grain; hence, one possibility is that precocious ger-mination would be prevented by the surrounding structures by impedingABA from leaching outside the embryo (Bewley and Black, 1994).

ABA content has been reported to peak at around PM and to decline af-terward when the grain undergoes desiccation (Goldbach and Michael,1976; Walker-Simmons, 1987; Quarrie, Tuberosa, and Lister, 1988; Morris,Jewer, and Bowles, 1991; Steinbach et al., 1995; Benech-Arnold et al.,1999). However, and in contrast to what might have been expected, no cor-relations have been found between ABA embryonic content during seed de-velopment and timing of exit from dormancy. In other words, although in-hibiting ABA synthesis (either genetically or through chemicals) has beenshown to accelerate the termination of dormancy, genotypes with a shortdormancy usually do not have lower ABA content during grain develop-ment than those with long-lasting dormancy. One exception for this lack ofcorrelation, however, has been reported for barley. In barley cultivars withcontrasting timing of exit from dormancy, ABA embryonic content is usu-ally similar until PM, and maximum ABA content also occurs prior to PM(Figure 5.2). However, immediately after PM, a dramatic reduction in em-bryonic ABA content takes place in embryos from the sprouting-suscepti-ble ‘B1215’, coinciding with the abrupt termination of hull-imposed dormancythat takes place in these grains after PM (Figure 5.1c); in ‘Q. Palomar’(a cultivar with longer-lasting dormancy) embryos, in contrast, ABA con-tent is kept at high levels for longer (i.e., until 43 DAP).

It has been suggested that dormancy imposed by the hull is mediated byhigh polyphenol-oxidase activity existing in the barley glumellae which re-sults in oxygen deprivation for the embryo (Lenoir, Corbineau, and Côme,1986). The way in which oxygen influences germination of dormant seedsis largely unknown, but it has been hypothesized that oxygen concentrationmight determine the rate with which germination inhibitors are catabolized(Neil and Horgan, 1987). This proposition is strongly supported by resultspresented by Wang and colleagues (1998) showing that the dormancybreaking effect of a strong oxidant such as hydrogen peroxide is through areduction in the endogenous level of the germination inhibitor abscisic acid.The question arising is, How can this mechanism operate differentiallythroughout development and between genotypes presenting different tim-ing of exit from dormancy? In the light of these results and within the frameof the proposition that the hull impedes embryo germination because it in-terferes with ABA oxidation (or metabolization) through oxygen depriva-tion, it could be argued that release from hull-imposed dormancy occurs be-cause oxygen in high concentrations is not necessary when germination

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inhibitors (i.e., ABA) are no longer present. These results explain the differ-ent timing of exit from dormancy between cultivars whose grains acquiregerminability immediately after PM or few days after harvest. However, inmost barley cultivars dormancy may last several months; in such cases, thecorrelation between ABA and germinability does not hold. Indeed, al-though inhibiting ABA synthesis with fluridone can anticipate exit fromdormancy, these cultivars do not present higher ABA content during graindevelopment and, on the other hand, ABA levels are barely detectable afterharvest maturity. Some authors have proposed that the maintenance of dor-mancy in those cultivars is mediated by de novo synthesis of ABA upongrain incubation, which would not occur in grains without dormancy (Wanget al., 1998). However, this possibility is still a subject of debate.

The role of changes in embryo responsiveness to ABA has been sug-gested to be a key one for controlling release from dormancy in cereals andother species (Robichaud, Wong, and Sussex, 1980; Walker-Simmons,

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FIGURE 5.2. Abscisic acid content (expressed as picograms of ABA per milli-gram of dry weight) in embryos from a sprouting-susceptible (‘B1215’, whitediamond) and sprouting-resistant (‘Quilmes Palomar’, black squares) cultivar,harvested at different days after pollination. PM and HM are the moments thecrops reached physiological and harvest maturity, respectively. (Source: Adaptedfrom figures in Benech-Arnold et al., 1999.)

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1987; Corbineau, Poljakoff-Mayber, and Côme, 1991; Steinbach et al.,1995; Benech-Arnold et al., 2000; Van Beckum, Libbenga, and Wang,1993; Wang et al., 1994; Wang, Heimovaara-Dijkstra, and Van Duijn, 1995;Visser et al., 1996). Embryo sensitivity to ABA is measured as the embryocapacity to overcome the inhibitory action of a certain concentration of thehormone. In the system ‘B1215’-‘Q. Palomar’ termination of hull-imposeddormancy is also correlated with changes in embryo sensitivity to ABA(Figure 5.3); release of ‘B1215’ grains from dormancy coincides with anabrupt loss of embryo sensitivity to ABA, while high responsiveness toABA is maintained for longer in ‘Q. Palomar’ embryos. Cultivars that havea lower embryo sensitivity to ABA during seed development usually pre-sent a faster release from dormancy. For example, a tenfold higher concen-tration of ABA is required to inhibit germination of embryos from a sorghumvariety whose grains are released from dormancy prior to PM than is neces-sary to inhibit germination of embryos from a variety with a long-lastingdormancy (Steinbach et al., 1995). The nature of the low sensitivity to ABAobserved in embryos from genotypes with short dormancy remains unclear,though some possibilities have been proposed. In an interesting paper,Visser and colleagues (1996) showed results suggesting that the low em-bryo sensitivity to ABA exhibited by a barley cultivar with no dormancywas not related to alterations in the ABA transduction pathway but to a highrate of degradation of the hormone in the outside walls of the embryo.

The Role of Gibberellins

The central role of gibberellins (GAs) in promoting seed germinationwas suggested decades ago and confirmed clearly since the identification ofGA-deficient mutants of Arabidopsis and tomato seeds that will not germi-nate unless exogenously supplied with GAs (Lona, 1956; Karssen et al.,1989; Hilhorst, 1995; Karssen, 1995). Similarly, dormant developing sor-ghum caryopses can be induced to germinate if incubated in the presence ofGAs (Steinbach, Benech-Arnold, and Sánchez, 1997). This role should notbe confounded with the postgerminative one referred to in Chapters 6 and13 when describing production of -amylase in barley and other germinat-ing grains. It has been proposed that endogenous GAs control germinationthrough two processes: a decrease in the mechanical resistance of the tis-sues surrounding the embryo and promotion of the growth potential of theembryo (see Chapter 7 for details; Bradford et al., 2000), thus antagonizingthe effect of ABA (Schopfer and Plachy, 1985). In cereals in which the tis-sues covering the embryo are weak or are split during imbibition, GA actionmust be restricted to promote embryo growth potential. Benech-Arnold and

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colleagues (2000) hypothesized that the low dormancy presented by devel-oping sorghum caryopses from sprouting-susceptible genotypes should beexpressed as a high capacity of the embryo to produce GA de novo synthe-sis upon grain imbibition; these GAs would be necessary to counterbalance

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FIGURE 5.3. Germination indexes (GI) of embryos from a barley cultivar with ashort dormancy (‘B1215’, ) and one with a longer lasting dormancy (‘Q. Palo-mar’, ), harvested at different days after pollination, after 12 days of incubationat 20°C, in the presence of 5 µM ABA (upper panel) or 50 µM ABA (lower panel).PM and HM are the moments the crops reached physiological and harvest matu-rity, respectively. (Source: Adapted from figures in Benech-Arnold et al., 1999.)

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the inhibitory effect imposed by the high ABA content existing during graindevelopment.

In addition to its role as germination promoter, it has been demonstratedthat the pattern of exit from dormancy in developing cereal grains can be al-tered by inhibiting GA synthesis, suggesting that this pattern depends onthe extent to which ABA action as a dormancy imposer is counterbalancedby the effect of GAs (Steinbach, Benech-Arnold, and Sánchez, 1997;Benech-Arnold et al., 1999). Applications of the GA synthesis inhibitorpaclobutrazol almost immediately after anthesis of barley and sorghum va-rieties with short dormancy results in a pattern of exit from dormancy thatresembles the characteristic pattern of varieties with a long-lasting dor-mancy, even though genotypes with a short dormancy have not been foundto present a lower GA content during development (Benech-Arnold et al.,2000). However, it could be predicted from experiments with paclobutrazolthat lowering GA content through genetic means should result in genotypeswith extended dormancy.

PHYSIOLOGY OF DORMANCY IN THE SUNFLOWER SEED

At harvest time sunflower seeds are dormant and germinate poorly(Corbineau, Bagniol, and Côme, 1990; Corbineau and Côme, 1987; Cse-resnyes, 1979). This dormancy is the result of true embryo dormancy(Corbineau, 1987; Corbineau, Bagniol, and Côme, 1990) and the inhibitoryaction of the envelopes (Corbineau, 1987; Corbineau, Bagniol, and Côme,1990; Corbineau and Côme, 1987) including the seed coat and the pericarpsince sunflower “seeds” are achenes.

The inception of embryo dormancy occurs relatively early throughoutseed development. Sunflower embryos are germinable if isolated from theentire seed from as early as 7 DAP and until approximately 12 DAP; the en-tire seed, however, germinates very poorly during this period, showing theexistence of coat (seed coat plus pericarp)-imposed dormancy (Figure 5.4)(LePage-Degivry and Garello, 1992; Corbineau, Bagniol, and Côme, 1990).From 12 DAP onward, embryo dormancy progressively develops, and at 20to 22 DAP, embryos are fully dormant (Figure 5.4). This embryo dormancyis not eliminated if the axis is separated from the cotyledons, indicating thatthe axis itself is dormant (LePage-Degivry and Garello, 1992). While theseed progresses toward maturation, embryos are slowly released from dor-mancy; by the time the grain has attained harvest maturity, some embryodormancy still persists (Figure 5.4). Therefore, the deep dormancy that sun-flower grains present at harvest results from the coexistence of coat-imposeddormancy and some remnant embryo dormancy (Corbineau, Bagniol, and

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Côme, 1990). Embryo dormancy is lost shortly after harvest if the seed issubjected to dry after-ripening, but coat-imposed dormancy persists for lon-ger and may require several weeks of dry after-ripening to be overcome.

The plant growth regulator ABA appears to be involved in the impositionof embryo dormancy. The inclusion of fluridone in culture media for sun-flower embryo development prevents the induction of embryo dormancy(LePage-Degivry, Barthe, and Garello, 1990; LePage-Degivry and Garello,1992). Nevertheless, the pattern of accumulation of ABA in the developingembryo does not coincide with the embryo physiological behavior. Duringseed development, embryos germinate well at the time when the endoge-nous ABA level is at its highest (7 to 12 DAP); thereafter, ABA decreases toa low value when embryo dormancy becomes established (LePage-Degivry,Barthe, and Garello, 1990). It seems, then, that the ABA peak at early stagesis responsible for the imposition of the dormant state that is established im-mediately after that peak has taken place.

Moreover, it appears that ABA needs to be present during a critical timeperiod to induce dormancy. In an interesting study, LePage-Degivry andGarello (1992) showed that when young (7 DAP), nondormant embryoswere cultured in the presence of ABA, the hormone produced a temporary

FIGURE 5.4. Germination percentage of sunflower achenes (solid triangles),naked seeds (open triangles), and isolated embryos (solid squares) harvested atdifferent days after pollination and incubated at 25 C. Whole achenes weretotally unable to germinate when incubated at any of the DAP displayed in thegraph.PM and HM are, approximately, the moments the crop reached physiolog-ical and harvest maturity, respectively. (Source: Redrawn with data from LePage-Degivry and Garello, 1992, and Corbineau, Bagniol, and Côme, 1990.)

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inhibition of germination but did not induce dormancy (i.e., embryos wereable to germinate when transferred to a basal medium). In contrast, exoge-nous ABA became effective if applied immediately prior to the natural in-duction of dormancy. For example, five days culture on a medium contain-ing 5 × 10–5 M ABA resulted in partial dormancy in 13 DAP embryos, whiletotal induction of dormancy occurred in 17 DAP embryos. The authors con-cluded that either a change in sensitivity to ABA occurs during develop-ment, or the existence of a second factor is necessary along with ABA to in-duce dormancy. Regarding this second possibility, the authors speculateabout the existence of a regulatory protein called VP that binds to ABA forthe induction of dormancy; ABA would not be able to induce dormancy in 7to 10 DAP embryos due to the absence of this protein. LePage-Degivry andGarello (1992) suggested that the capacity of the embryo to produce in situABA synthesis, which appears during seed development along with the on-set of dormancy, is necessary not only to induce, but also to maintain dor-mancy.

As mentioned previously, embryo dormancy can be terminated by drystorage. Bianco, Garello, and LePage-Degivry (1994) attempted to eluci-date the mechanism through which dry storage terminates embryo dor-mancy by drying artificially dormant 17 to 26 DAP embryos and testing forgerminability either immediately after drying or after leaving the embryosfor six weeks in a desiccator (dry storage). They observed a decrease inABA content immediately after the drying process that was not accompa-nied by a complete release from dormancy. On the other hand, additionaldry storage did not affect the ABA content but instead promoted germina-tion. In addition, the authors found that the drying treatment also stimulatedimmature sunflower embryos and axes to respond to gibberellins uponrehydration. The authors concluded from these results that although thedrying treatment induced both a decline in ABA and an increase in sensitiv-ity to GA, additional dry storage is necessary to obtain germination. Theypropose the suppression induced during this dry storage of the aforemen-tioned capacity to produce in situ ABA synthesis in the embryo, as themechanism behind the response, though they did not show the extent towhich the drying treatment by itself could also result in such suppression.

The inception of seed coat plus pericarp-imposed dormancy occurs earlythroughout seed development: by the stage at which young (7 to 13 DAP),nondormant embryos can germinate readily if isolated from the entire seed,germination of the whole grain is prevented by the presence of the enve-lopes (Figure 5.4). Coat-imposed dormancy possibly continues during therest of the developmental period, but its existence is difficult to corroboratebecause the embryo itself is dormant during most of this period. Once theseed has completed maturation and while the embryo gradually loses its

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dormancy, coat-imposed dormancy persists for longer, in some cases forseveral months.

The nature of this inhibition imposed on embryo germination is highlyunknown, though it has been suggested that both pericarp and seed coat in-terfere with oxygen difussion toward the embryo (Gay, Corbineau, andCôme, 1991). As in the case of hull-imposed dormancy in barley, it could bespeculated that the envelope impedes embryo germination because it in-terferes with ABA and/or other inhibitor oxidation (or metabolization)through oxygen deprivation. Similarly, the mechanism through which dryafter-ripening alleviates coat-imposed dormancy has not been explored tothe best of our knowledge. It could be that, even after the embryo has beenreleased from dormancy, it retains the capacity to produce ABA synthesisupon imbibition, which might be necessary to maintain (coat-imposed) dor-mancy; indeed, oxygen deprivation caused by the presence of the embryowould prevent ABA oxidation. If, as mentioned before, dry storage sup-presses the capacity of the embryo to produce ABA synthesis (Bianco,Garello, and LePage-Degivry, 1994), then coat-imposed dormancy wouldbe terminated because oxygen in high concentrations should not be neces-sary when ABA is no longer present. This hypothesis should be thoroughlytested. Unfortunately, we are not aware of any study in which the physiol-ogy of dormancy in sunflower has been comparatively investigated in geno-types with contrasting duration of dormancy.

THE EXPRESSION OF DORMANCY IN GRAIN CROPS

Except for the case of seeds that present full dormancy and consequentlydo not germinate at either temperature, it is a common feature that dor-mancy is expressed at certain temperatures. Vegis (1964) introduced theconcept of degrees of relative dormancy from the observation that, as dor-mancy is released, the temperature range permissive for germination wid-ens, until germination is maximal under a wide thermal range. This is alsothe case for dormant cereal grains: in summer cereals such as sorghum, dor-mancy is not expressed at high temperatures (i.e., 30ºC) (Benech-Arnold etal., 1995; Benech-Arnold, Enciso, and Sánchez, 1999), and in winter cere-als such as wheat and barley it is not expressed at low temperatures (i.e.,10ºC or lower) (Bewley and Black, 1994; Gosling et al., 1981; Mares, 1984;Black, Butler, and Hughes, 1987; Walker-Simmons, 1988). It should be em-phasized that the depressed germination which occurs as temperatures ex-ceed (in the case of winter cereals) or are below (in the case of summer cere-als) certain values is truly an expression of dormancy and not an inevitableeffect of temperature on germination, for it does not take place in isolated

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embryos or in grains which have after-ripened (Mares, 1984). Moreover, ithas been shown in wheat that isolated embryos incubated at high tempera-tures (i.e., 25 to 30ºC) are more effectively inhibited by ABA than embryosincubated at lower temperatures (Walker-Simmons, 1988). This thermalrange permissive for germination widens with after-ripening so grains be-come able to germinate at most temperatures. Similarly, it was observed forbarley grains that, so long they are released from dormancy throughout de-velopment and maturation, they are able to germinate at higher tempera-tures (Benech-Arnold, Enciso, and Sánchez, 1999). This differential ex-pression of dormancy which depends on the incubation temperature alsohas implications for crop behavior in the field. For example, the lack of ex-pression of dormancy at low temperatures, characteristic of winter cereals,implies that in years when damp conditions are combined with low air tem-peratures around harvest time, both resistant (high dormancy) and suscepti-ble (low dormancy) cultivars might be expected to sprout. The inverse couldbe said about summer cereals such as sorghum; high temperatures com-bined with damp conditions around harvest permit the germination inplanta of both dormant and nondormant cultivars.

The amount of water in the incubation medium also allows differentialexpression of dormancy in barley grains. Indeed, most barley cultivarswhich present some dormancy at harvest will not germinate if the grains areincubated in a petri dish at favorable temperatures but with 8 or even 6 mlinstead of 4 ml of distilled water (Pollock, 1962); the same does not occur ingrains from cultivars with low dormancy, or in those that have after-ripened,showing that it is truly an expression of dormancy (Figure 5.5). This phe-nomenon is known by the malting industry as “sensitivity to water” and isone of the quality parameters assessed upon reception of a grain lot. Thissensitivity to water must be related to the oxygen deprivation imposed bythe presence of the hull, described previously, which might be enhanced bythe hypoxia that results from an excess of water in the incubation media.

In the case of freshly harvested sunflower seeds, dormancy is expressedat temperatures lower and higher than 25ºC (Corbineau, Bagniol, andCôme, 1990). Dormancy expression at low temperatures is attributed to em-bryo dormancy which is not expressed at high temperatures (Corbineau,Bagniol, and Côme, 1990); conversely, dormancy expressed at high tem-peratures results from coat-imposed dormancy (Corbineau, Bagniol, andCôme, 1990). Consequently, a few weeks of dry after-ripening allows seedgermination at low temperatures due to termination of embryo dormancy;the acquisition of the capacity to germinate at high temperatures, in con-trast, may take several weeks of dry after-ripening (Corbineau, Bagniol, andCôme, 1990).

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REMOVING DORMANCY AT AN INDUSTRIAL SCALE

In some cases it is not possible to wait for the effect of dry after-ripeningto take place and termination of grain dormancy must be anticipated. This isfrequently the case with malting barley, whose germination is required forindustrial utilization (see Chapter 13) and also with sunflower whose grainsare usually dormant by the time they are needed for generating a new crop.

One of the most popular methods used by the malting industry, when-ever allowed by the customer, is the addition of gibberellic acid (GA3) to theincubation medium to promote the germination of dormant barley grains.Indeed, it is well known that gibberellic acid at low concentrations (0.1 to0.2 ppm) stimulates germination in these grains (Brookes, Lovett, andMacWilliam, 1976). Studies on the most appropriate point in the malting

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FIGURE 5.5. Response of barley germination to the amount of water present:(A) freshly harvested dormant barley; (B) the same barley, still water sensitivethough not fully dormant; (C) the same barley after some time of after-ripening indry storage. (Source: Adapted from a figure in Pollock, J.R.A., 1962.)

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process at which to add gibberellic acid have concluded that it should besprayed on soon after the grain is removed from the steep (Brookes, Lovett,and MacWilliam, 1976). Other methods to remove dormancy in barley in-clude the use of dilute solutions of hydrogen sulfide and keeping the grainsfor three days at 40ºC, either in the open air when their moisture content fellto about 8 percent, or in closed vessels, when moisture contents were un-changed at between 17 and 20 percent (Pollock, 1962).

As with other cultivated species such as Lactuca sativa (Abeles, 1986)and Arachis hypogaea (Ketring, 1977), ethylene (C2H4) and etephonstrongly stimulate the germination of dormant sunflower seeds (Srivastasaand Dey, 1982; Corbineau and Côme, 1987; Corbineau, Bagniol, andCôme, 1990). In contrast, gibberellic acid and cold stratification do notovercome dormancy in this species (Bagniol, 1987) though it was shownthat 1 mM GA3 is effective for overcoming dormancy in some wild sun-flowers (Seiler, 1998). Corbineau, Bagniol, and Côme (1990) showed thatethylene and its immediate precursor (1-aminocyclopropane-1-carboxylicacid) strongly stimulated germination of primary dormant sunflower seeds;on the contrary, inhibitors of ethylene (i.e., amino-oxyacetic acid andCoCl2) or ethylene action (silver thiosulfate and 2.5 norbomadiene) inhib-ited germination of nondormant seeds. Beyond the evident practical impli-cations of these results, they also indicate that ethylene synthesized by theseeds themselves is involved in the regulation of sunflower seed germina-tion. The use of ethylene or its precursors appears as a promising technol-ogy to stimulate the germination of dormant sunflower lots. Possibly, seedcompanies have not adopted it yet, due to the inexistence of adequate de-vices to treat large amounts of seeds.

GENETICS AND MOLECULAR BIOLOGYOF DORMANCY IN GRAIN CROPS

Although some investigations indicate that dormancy is controlled byone or two recessive genes (Bhatt, Ellison, and Mares, 1983), in severalstudies, seed dormancy has been revealed as a quantitative trait (i.e., a traitwith continuous phenotypic variation). Consequently, modern approachesfor determining the genetic bases of seed dormancy include the use of mo-lecular markers (AFLP [amplified fragment length polymorphism] or RFLP[restriction fragment length polymorphism]) to identify QTLs (quantitativetrait loci) or, in other words, loci controlling the quantitative trait “dor-mancy.” Wheat had three QTLs that explained more than 80 percent of thetotal phenotypic variance in seed dormancy (Kato et al., 2001). A majorQTL was located on the long arm of chromosome 4A, and two minor QTLs

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were on chromosomes 4B and 4D. In sorghum, two unlinked QTL, phsEand phsF, were found to influence dormancy in an F2 population generatedby the cross of a sprouting-susceptible variety with a sprouting-resistantone. These two QTLs accounted together for 53 percent of the phenotypicvariance for preharvest sprouting (Lijavetzky et al., 2000).

Early genetic investigations (Buraas and Skinnes, 1984) revealed thatseed dormancy in Scandinavian barleys was governed by several recessive,nucleoplasmic loci with high heritability. Genetic control of barley seeddormancy has also been studied by means of QTL mapping (Ullrich et al.,1993; Takeda, 1996). A saturated molecular marker linkage map based onthe six-row Steptoe/Morex (S/M) mapping population has been developed(Kleinhofs et al., 1993) and extensively used for QTL analysis by the NorthAmerican Barley Genome Mapping Project (Hayes et al., 1993; Han et al.,1996; Romagosa et al., 1996). Steptoe is a six-row feed barley with highlevels of dormancy (Muir and Nilan, 1973). Morex is a six-row malting typethat does not express dormancy (Rasmusson and Wilcoxson, 1979). Fourregions of the barley genome on chromosomes 1 (7H), 4 (4H), and 7 (5H)were associated with most of the differential genotypic expression for dor-mancy in the S/M cross (Ullrich et al., 1996; Oberthur et al., 1995; Hanet al., 1996; Larson et al., 1996). They were designated SD1 to SD4 by Hanand colleagues (1996) and accounted for approximately 50, 15, 5, and 5percent of the phenotypic differences, respectively, in germination follow-ing several post-harvest periods. In an early study, Livers (1957; cited byRomagosa et al., 1999) found some evidence that one or more postharvestdormancy (phd) genes may be located on chromosome 7, which is wheretwo of the S/M QTLs are located. Takeda (1996), using QTL analysis withthe Harrington/TR306 (H/T), population identified one region each onchromosomes 5 (1H) and 7 (5H) that controlled dormancy. The chromo-some 7 (5H) H/T QTL coincides with the S/M QTL SD2 on the end of thelong arm and was suggested to be allelic. In a recent study, Romagosa andcolleagues (1999) investigated the individual effects on the S/M SD QTL ondormancy during seed development and after-ripening. With this aim, threepairs each of doubled haploid lines (DHLs) derived from Steptoe/MorexF1s with the MM SS, SS MM, and SS SS genotypes at the SD1 and SD2QTL and fixed M genotypes (MM MM) at the SD3 and SD4 QTL wereidentified by RFLP analysis. Morex and genotype MM SS MM MM werethe first to start losing dormancy throughout development; the other geno-types remained dormant until the end of seed development (Figure 5.6a).Similarly, Morex and genotypes MM SS MM MM and MM SS SS SS hadcompletely lost dormancy after 30 days of after-ripening, while other geno-types presented a pattern of exit from dormancy which progressively resem-bled that observed for the highly dormant Steptoe (Figure 5.6b). Since the

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FIGURE 5.6. Germination percentage of various barley genotypes during seeddevelopment (A) and during after ripening after crop harvest (B). Genotypicmeans followed by the same letter are not statistically significant according toDuncan test ( < 0.05) (a) or LSD test ( < 0.05) (b) MM (Morex) and SS(Steptoe) designation refer to the genotypes of the pair of flanking markers forthe four seed dormancy (SD) QTLs in order: SD1, SD2, SD3, SD4, e.g., MM SSMM MM. (Source: From Romagosa et al., 1999. Reproduced with permission.)

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presence of the Steptoe allele at SD1 on chromosome 7 (5H) delayed exitfrom dormancy, the authors concluded that SD1 is the most important QTLin determining the time of dormancy release.

We are not aware of any study carried out to identify the genetic basis ofdormancy in the sunflower crop.

Although work with molecular markers is extremely valuable, studieslinking the molecular biology with the physiology (i.e., identification ofcandidate genes) appear to be a promising means of achieving the objectiveof adjusting release from dormancy to a precise and narrow time window as,for example, is required in the case of barley. The gene Vp1 encodes a tran-scription factor whose involvement in the control of embryo sensitivity toABA has been evidenced since the isolation of maize vp1 mutants that areinsensitive to ABA and present viviparity (McCarty et al., 1991). Preharvestsprouting in cereals is very similar phenotypically to the vp1 mutation inmaize, raising the interesting possibility that preharvest sprouting in barleyand other cereals is caused, in part, by the physiological disruption of theVp1 function. Genes homologous to Vp1 from barley (Hollung et al., 1997)and other Gramineae such as rice (Hattori, Terada and Hamasuna, 1994),sorghum (Carrari et al., 2001), and Avena fatua (Jones, Peters, and Holds-worth, 1997) have been cloned and sequenced, and, in some cases, closecorrelations between Vp1 expression and dormancy were found (Jones, Pe-ters, and Holdsworth, 1997). In other cases, however, such a correlation wasnot found. Carrari and colleagues (2001), using two sorghum varieties withcontrasting duration of dormancy, did not see any straightforward relation-ship between Vp1 expression during seed development and the particularpattern of exit from dormancy. In other words, the expression levels of Vp1during development cannot predict the future germination behavior of theimmature seed upon imbibition. Moreover, Vp1 has recently been mappedusing the Redland B2/IS 9530 system used by Lijavetzky and colleagues(2000) to identify QTLs controlling dormancy in sorghum. Vp1 did not mapwithin any of these QTLs (Lijavetzky et al., 2000). Nevertheless, McKibbinand colleagues (2002) have recently analyzed Vp1-transcript structure inwheat embryos during grain development and found that a homeologueproduces cytoplasmic mRNAs of different size. They observed that the ma-jority of transcripts are spliced incorrectly, contain insertions of intron se-quences or deletions of coding region, and do not have the capacity to en-code full-length proteins. These authors suggest that missplicing of wheatVp1 genes contributes to an early release of dormancy of the grains whichfrequently results in preharvest sprouting (McKibbin et al., 2002). In con-trast, Avena fatua Vp1 genes do not show the same missplicing and, inagreement with the idea that Vp1 gene exerts control on dormancy, A. fatuagrains present a persistent dormancy. Interestingly, developing embryos

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from transgenic wheat grains expressing the Avena fatua Vp1 showed en-hanced responsiveness to applied ABA, and ripening ears were less suscep-tible to preharvest sprouting (McKibbin et al., 2002). These results, then,identify a possible route to manipulate dormancy duration in wheat.

Protein kinases often act in the transduction of external signals and couldhave a role in the effects of environmental conditions on expression of dor-mancy. For that reason a protein kinase mRNA (PKABA1) that accumu-lates in mature wheat seed embryos and that is responsive to applied ABAwas cloned and its expression analyzed during imbibition of dormant andnondormant wheat seeds. When dormant seeds are imbibed, embryonicPKABA1 mRNA levels remain high for as long as the seeds are dormant,while they decline and disappear in embryos of germinating seeds (Ander-berg and Walker-Simmons, 1991; Holappa and Walker-Simmons, 1995).The role of this kinase in dormant seeds is currently under investigation, buta potential role of phosphorylation-dependent responses in maintenance ofseed dormancy is also supported by characterization of the abi1 mutant ofArabidopsis (an ABA-insensitive with no dormancy) (Meyer, Leube, andGrill, 1994). The participation of this protein kinase in maintaining dor-mancy in grains from other crops remains to be investigated. G-protein-coupled receptors can participate in hormone-dependent signaling cascadesaffecting germination-related genes. Recently it has been shown that over-expression of GCR1, a G-protein-coupled receptor gene, decreases seeddormancy in Arabidopsis (Colucci et al., 2002). Whether expression of thistype of gene is related to dormancy depth in cereal seeds is not yet known,although it is an interesting possibility.

Differences in gene expression in imbibed dormant and nondormantcaryopses of Avena fatua (wild oats) have been determined through thetechnique of differential display (Li and Foley, 1994, 1995; Johnson et al.,1995). Monitoring gene expression in dormant and nondormant caryopsesof barley through differential display could eventually evince yet unknownphysiological and biochemical mechanisms controlling dormancy, pro-vided the function of genes that are differentially expressed is finally eluci-dated.

In summary, both genetics and molecular biology studies could aid in thesearch for cultivars that, without having a long-lasting dormancy, couldpresent resistance to preharvest sprouting. However, the complementaritywith physiological studies is essential if such a goal is to be attained. Themost profitable genetic investigations would be those that, for example,through QTL analysis, demonstrate the participation of genes with knownphysiological function. If in the end the phenotype happens to correlatewell with some characteristic of that gene (i.e., differences between pheno-

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types in terms of gene expression timing, sequence, regulation, etc.) thenthe possibilities for manipulating the system are high.

ENVIRONMENTAL CONTROL OF DORMANCYIN GRAIN CROPS

The duration of dormancy is determined mainly by the genotype, but, asin many other species, it is known that dormancy in grain crops can also beinfluenced by the environment experienced by the mother plant (Kahn andLaude, 1969; Nicholls, 1982; Reiner and Loch, 1976; Schuurink, VanBeckum, and Heidekamp, 1992; Cochrane, 1993; see also Auranen, 1995,for references). Indeed, the effects of the parental environment on seed dor-mancy have been reported for a wide range of species (for reviews, seeFenner, 1991; Wulff, 1995). Some well-defined patterns emerge, however,with certain environmental factors tending to have similar effects in differ-ent species. For example, low dormancy is generally associated with hightemperatures, short days, drought and nutrient availability during seed de-velopment (Walker-Simmons and Sesing, 1990; Fenner 1991; Benech-Arnold, Fenner, and Edwards, 1991, 1995; Gate, 1995). The assessmentand quantification of these effects might lead to the development of predic-tive models that could be of great help for reducing the incidence of prob-lems derived from either a short or a persistent dormancy.

Among the different factors acting on the mother plant, temperature ap-pears to be primarily responsible for year-to-year variation in grain dor-mancy within a genotype. Evidence suggests that temperature might be ef-fective only within a sensitivity period during grain filling (Kivi, 1966;Reiner and Loch, 1976; Buraas and Skinnes, 1985). Reiner and Loch(1976) determined that low temperatures during the first half of grain fill-ing, combined with high temperatures during the second half, result in a lowdormancy level of the barley grain and, presumably, in preharvest sproutingsusceptibility. Conversely, high temperatures during the first half combinedwith low temperatures during the second produced the highest dormancylevels. The authors established a linear relationship between the ratio of thetemperatures prevailing at both halves of the filling period and the dor-mancy level of the grains three weeks after harvest. This model has sincebeen used by the German malting industry to predict dormancy level in themalting barley harvest lots.

In a recent work, Rodriguez and colleagues (2001) identified a time win-dow within the grain-filling period of cultivar Quilmes Palomar with sensi-tivity to temperature for the determination of dormancy. This time windowwas found to occur a few days before physiological maturity (PM). Specifi-

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cally, the duration of the phase heading PM for this cultivar was determinedto last, on a thermal time scale, 420ºC days (accumulated over a base tem-perature of 5.5ºC). The sensitivity window was found to start at 300ºC dayafter heading and to finish at 350ºC day after heading. A positive linear rela-tionship was established between the average temperature perceived by thecrop during this time window and the germination index of the grains 12days after PM (Figure 5.7).

Twelve days after PM is approximately halfway between physiologicaland harvest maturity; grain germination index measured at this stage is agood estimate of the rate at which the grains are being released from dor-mancy after PM. According to this model, the higher the temperature expe-rienced during the sensitivity time window, the faster the rate with whichgrains will be released from dormancy after PM and, consequently, thelower the dormancy level prior to crop harvest. Such a situation, combinedwith a forecast of heavy rains for the forthcoming days, implies a risk forthe crop and the farmer could decide to harvest before the crop has reachedfull maturity. Conversely, low temperatures experienced by the crop duringthe sensitivity window would result in a high dormancy level prior to har-vest, making the crop resistant to sprouting. This model was successfullyvalidated with data collected from commercial plots 700 km away from the

FIGURE 5.7. The relationship between temperature experienced by the crop inthe sensitivity window going from 300 to 350°C days after heading (Tm300-350),and the germination index of grains harvested 12 days after physiological matu-rity (GI [12 DAPM]) and incubated at 20°C. (Source: Redrawn with data from Ro-driguez et al., 2001.)

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site where the model was produced (Rodriguez et al., 2001). However, itwas also noted that temperature explains only one dimension of the ob-served variability in dormancy. Indeed, some other unknown factors wereresponsible for influencing the relationship between temperature and dor-mancy (Rodriguez et al., 2001). Current efforts are directed toward identi-fying these factors and quantifying their effects.

As an exception to the general rule stating that low dormancy is gener-ally associated with high temperatures experienced during grain filling, ithas been found for sunflower that high temperatures during grain develop-ment result in an extended period of dormancy (Fonseca and Sánchez,2000). In this case, germination was tested at low incubation temperatures(i.e., grains that had developed at high temperatures required longer time ofdry after-ripening to acquire the capacity to germinate at low temperatures).Since embryo dormancy is expressed at low temperatures, it might be, then,that high temperatures during grain filling extended the duration of embryodormancy. Germination at high temperatures, however, was not tested, andtherefore it is not possible to say whether the duration of seed coat dor-mancy was also extended by high temperatures during grain development.

CONCLUDING REMARKS

The task of adjusting the timing of exit from dormancy of grain crops tothe needs of both farmers and industry does not seem to be an easy one.However, an adequate knowledge of the physiology and the genetics of dor-mancy in grain crops should help to solve the conflict between obtainingcultivars with low dormancy at harvest but not with such an anticipated ter-mination of dormancy that leads to sprouting. Although much progress hasbeen made in recent years, we are still far away from having detailed knowl-edge on the physiology and genetics of dormancy. It is worth emphasizingthat studies linking the genetics (and the molecular biology) with the physi-ology appear to be the most promising ones. For example, if genes control-ling sensitivity to hormones (either ABA or GAs) are finally identified andtheir participation in the control of dormancy is eventually evidenced, thenefforts should be directed to understand the regulation of those genes. Itwould not be surprising to find out that the action of genes controlling, forexample, sensitivity to ABA, is cancelled after the grain has undergone des-iccation (Kermode, 1995). If the transduction pathway is finally under-stood, then it should not be very difficult to manipulate the timing of suchcancellation. This is just one example to illustrate how molecular studiesoriented by physiological studies could yield tools for the production of ge-notypes with a precise timing of dormancy release.

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Our knowledge on how the environment modulates the timing of exitfrom dormancy in grain crops could also help to make management decisionsto reduce the incidence of problems derived from dormancy. Throughoutthis chapter it was described how a comprehensive assessment of the effectsof temperature on dormancy during seed development can be used for de-ciding management practices. It is quite evident, however, that other factorsin addition to temperature modulate the timing of exit from dormancy.When these factors are identified and their effects quantified, decisions onmanagement practices will be made on an even more solid basis.

REFERENCES

Abeles, F.B. (1986). Role of ethylene in Lactuca sativa cv. Grand Rapids seed ger-mination. Plant Physiology 81: 780-787.

Anderberg, R.J. and Walker-Simmons, M.K. (1991). Isolation of a wheat cDNAclone for an abscisic acid-inducible transcript with homology to protein kinases.Proceedings of the National Academy of Sciences, USA 89: 10183-10187.

Auranen, M. (1995). Pre-harvest sprouting and dormancy in malting barley innorthern climatic conditions. Acta Agriculturae Scandinavica 45: 89-95.

Bagniol, S. (1987). Mise en évidence de l’intervention de l’ethylene dans la germi-nation et la dormance des semences de tournesol (Helianthus annuus L.).Diplôme d`Ëtudes Approfondies. Université Pierre et Marie Curie, Paris.

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Benech-Arnold, R.L., Enciso, S., Sánchez, R.A., Carrari, F., Perez-Flores, L.,Iusem, N., Steinbach, H.S., Lijavetzky, D., and Bottini, R. (2000). Involvementof ABA and GAs in the regulation of dormancy in developing sorghum seeds. InBlack, M., Bradford, K.J., and Vázquez Ramos, J. (Eds.), Seed Biology: Ad-vances and Applications (pp. 101-111). Oxon, UK: CAB International.

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Sorghum varieties with contrasting pre-harvest sprouting susceptibility. Journalof Experimental Botany 46: 711-717.

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Carrari, L., Perez-Flores, J., Lijavetzky, D., Enciso, S., Sanchez, R.A., Benech-Arnold, R.L., and Iusem, N. (2001). Cloning and expression of a sorghum genewith homology to maize vp1: Its potential involvement in pre-harvest sproutingresistance. Plant Molecular Biology 45: 631-640.

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Corbineau, F. and Côme, D. (1987). Regulation de las semences de tournesol parl’éthylene. In Annales ANPP, 2ème Colloque sur les substances de croissance etleurs utilisations en agriculture, Volume 1 (pp. 271-282). Paris: AssociationNationale de Protection des Plantes.

Corbineau, F., Poljakoff-Mayber, A., and Côme, D. (1991). Responsiveness toabscisic acid of embryos of dormant oat (Avena sativa) seeds: Involvement ofABA-inducible proteins. Physiologia Plantarum 83: 1-6.

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Hattori, T., Terada, T., Hamasuna, S.T. (1994). Sequence and functional analyses ofthe rice gene homologous to the maize Vp1. Plant Molecular Biology 24: 805-810.

Hayes, P.M., Liu, B.H., Knapp, S.J., Chen, F., Jones, B., Blake, T., Franckowiak, J.,Rasmusson, D., Sorrells, M., Ullrich, S.E., et al. (1993). Quantitative trait locuseffects and environmental interaction in a sample of North American barleygermplasm. Theoretical and Applied Genetics 87: 392-401.

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Holappa, L.D. and Walker-Simmons, M.K. (1995). The wheat abscisic acid-respon-sive protein kinase mRNA, PKABA1, is up-regulated by dehydration, cold tem-perature and osmotic stress. Plant Physiology 108: 1203-1210.

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Johnson, R.R., Cranston, H.J., Chaverra, M.E., and Dyer, W.E. (1995). Character-ization of cDNA clones for differently expressed genes in embryos of dormantand nondormant Avena fatua L. caryopses. Plant Molecular Biology 28: 113-122.

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Karssen, C.M. (1995). Hormonal regulation of seed development, dormancy, andgermination studied by genetic control. In Kigel, J. and Galili, G. (Eds.), SeedDevelopment and Germination (pp. 333-350). New York: Marcel Dekker, Inc.

Karssen, C.M., Brinkhorst-Van der Swan, D.L.C., Breekland, A.E., and Koorneef,M. (1983). Induction of dormancy during seed development by endogenousabscisic acid: Studies on abscisic acid deficient genotypes of Arabidopsis thali-ana (L.). Planta 157: 158-165.

Karssen, C.M., Zagorski, S., Kepczynski, J., and Groot, S.P.C. (1989). Key role forendogenous gibberellins in the control of seed germination. Annals of Botany 63:71-80.

Kato, K., Nakamura, W., Tabiki, T., Mura, H., and Sawada, S. (2001). Detection ofloci controlling seed dormancy on group 4 chromosomes of wheat and compara-tive mapping with rice and barley genomes. Theoretical and Applied Genetics102: 980-985.

Kermode, A.R. (1995). Regulatory mechanisms in the transition from seed develop-ment to germination: Interactions between the embryo and the seed environ-ment. In Kigel, J. and Galili, G. (Eds.), Seed Development and Germination (pp.273-332). New York: Marcel Dekker, Inc.

Ketring, D.L. (1977). Ethylene and seed germination. In Khan, A.A. (Ed.), ThePhysiology and Biochemistry of Seed Dormancy and Germination (pp. 157-178). Amsterdam: Elsevier, North Holland Biomedical Press.

Khan, R.A. and Laude, H.M. (1969). Influence of heat stress during seed maturationon germinability of barley seed at harvest. Crop Science 9: 55-58.

King, R.W. (1982). Abscisic acid in seed development. In Khan, A.A. (Ed.), ThePhysiology and Biochemistry of Seed Dormancy and Germination (pp. 157-181). Amsterdam: Elsevier, North Holland Biomedical Press.

Kivi, E. (1966). The response of certain pre-harvest climatic factors on sensitivity tosprouting in the ear of two-row barley. Acta Agriculturae Fennica 107: 228-246.

Kleinhofs, A., Kilian, A., Saghai Maroof, M.A., Biyashev, R.M., Hayes, P.M.,Chen, F.Q., Lapitan, N., Fenwich, A., Blake, T.K., Kanazin, V., et al. (1993). Amolecular, isozyme and morphological map of the barley (Hordeum vulgare) ge-nome. Theoretical and Applied Genetics 86: 705-712.

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Larson, S., Bryan, G., Dyer, W., and Blake, T. (1996). Evaluating gene effects of amajor barley seed dormancy QTL in reciprocal backcross populations. Journalof Agricultural Genomics 2: Article 4. Available at <http://www.cabi-publishing.org/gateways/jag/index.html>.

Le Page-Degivry, M.T., Barthe, P., and Garello, G. (1990). Involvement of endoge-nous abscisic acid in onset and release of Helianthus annuus embryo dormancy.Plant Physiology 92: 1164-1168.

Le Page-Degivry, M.T. and Garello, G. (1992). In situ abscisic acid synthesis: A re-quirement for induction of embryo dormancy in Helianthus annuus. Plant Phys-iology 98: 1386-1390.

Lenoir, C., Corbineau, F., and Côme, D. (1986). Barley (Hordeum vulgare) seeddormancy as related to glumella characteristics. Physiologia Plantarum 68: 301-307.

Li, B. and Foley, M.E. (1994). Differential polypeptide patterns in imbibed dormantand after-ripened Avena fatua embryos. Journal of Experimental Botany 45:275-279.

Li, B. and Foley, M.E. (1995). Cloning and characterization of differentially ex-pressed genes in imbibed dormant and after-ripened Avena fatua embryos. PlantMolecular Biology 29: 823-831.

Lijavetzky, D., Martinez, M.C., Carrari, F., and Hopp, H.E. (2000). QTL analysisand mapping of pre-harvest sprouting resistance in Sorghum. Euphytica 112:125-135.

Livers, R.W. (1957). Linkage studies with chromosomal translocation stocks in bar-ley. PhD Thesis (Diss. Abstr. AAT 5801125). St. Paul: University of Minnesota.

Lona, F. (1956). L’acido gibberéllico determina la germinazione del semi di Lac-tuca scariola in fase di scotoinhibizione. Ateneo Pamense 27: 641-644.

Mares, D. (1984). Temperature dependence of germinability of wheat (Triticumaestivum) grain in relation to pre-harvest sprouting. Australian Journal of Agri-cultural Research 35: 115-128.

McCarty, D.R., Hattori, T., Carson, C.B., Vasil, V., Lazar, M., and Vasil, I.K.(1991). The viviparous-1 developmental gene of maize encodes a novel tran-scriptional activator. Cell 66: 895-905.

McKibbin, R.S., Wilkinson, M.D., Bailey, P.C., Flintham, J.E., Andrew, L.M.,Lazzeri, P.A., Gale, M.D., Lenton, J.R., and Holdsworth, M.J. (2002). Tran-scripts of Vp-1 homeologues are misspliced in modern wheat and ancestral spe-cies. Proceedings of the National Academy of Sciences 99: 10203-10208.

Meyer, K., Leube, M.P., and Grill, E. (1994). A protein phosphatase 2C involved inABA signal transduction in Arabidopsis thaliana. Science 264: 1452-1455.

Morris, P.C., Jewer, P.C., and Bowles, D.J. (1991). Changes in water relations andendogenous abscisic acid content of wheat and barley grains and embryos duringdevelopment. Plant, Cell and Environment 14: 443-446.

Muir, C.E. and Nilan, R.A. (1973). Registration of Steptoe barley. Crop Science 13:770.

Neil, S.J. and Horgan, R. (1987). Abscisic acid and related compounds. In Rivier, L.and Crozier, A. (Eds.), The Principles and Practice of Plant Hormone Analysis(pp. 111-167). London: Academic Press.

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Nicholls, P.B. (1982). Influence of temperature during grain growth and ripening ofbarley on the subsequent response to exogenous gibberellic acid. AustralianJournal of Plant Physiology 9: 373-383.

Norstog, K. and Klein, R.M. (1972). Development of cultured barley embryos: II.Precocious germination and dormancy. Canadian Journal of Botany 50: 1887-1894.

Oberthur, L., Dyer, W., Ullrich, S.E., and Blake, T.K. (1995). Genetic analysis ofseed dormancy in barley (Hordeum vulgare L.) Journal of Agricultural Geno-mics 1: Article 5. Available at <http://www.cabi-publishing.org/gateways/jag/index.html>.

Pollock, J.R.A. (1962). The nature of the malting process. In Cook, A.M. (Ed.), Bar-ley and Malt: Biology Biochemistry, Technology (pp. 303-398). New York: Aca-demic Press.

Quarrie, S.A., Tuberosa, R., and Lister, P.G. (1988). Abscisic acid in developinggrains of wheat and barley genotypes differing in grain weight. Plant GrowthRegulation 7: 3-17.

Rasmusson, D.C. and Wilcoxson, R.D. (1979). Registration of Morex barley. CropScience 19: 293.

Reiner, L. and Loch, V. (1976). Forecasting dormancy in barley—Ten years experi-ence. Cereal Research Communication 4: 107-110.

Robichaud, C.S., Wong, J., and Sussex, I.M. (1980). Control of in vitro growth ofviviparous embryo mutants of maize by abscisic acid. Developmental Genetics1: 325-330.

Rodriguez, V., González Martín, J., Insausti, P., Margineda, J.M., and Benech-Arnold, R.L. (2001). Predicting pre-harvest sprouting susceptibility in barley:A model based on temperature during grain filling. Agronomy Journal 93: 1071-1079.

Romagosa, I., Han, F., Clancy, J.A., and Ullrich, S.E. (1999). Individual locus ef-fects on dormancy during seed development and after ripening in barley. CropScience 39: 74-79.

Romagosa, I., Ullrich, S.E., Han, F., and Hayes, P.M. (1996). Use of additive maineffects and multiplicative interaction model in QTL mapping for adaptation inbarley. Theoretical and Applied Genetics 93: 30-37.

Schopfer, P. and Plachy, C. (1985). Control of seed germination by abscisic acid:III. Effect on embryo growth potential (minimum turgor pressure) and growthcoefficient (cell wall extensibility) in Brassica napus L. Plant Physiology 77:676-686

Schuurink, R.C., Van Beckum, J.M.M., and Heidekamp, F. (1992). Modulation ofgrain dormancy in barley by variation of plant growth conditions. Hereditas 117:137-143.

Seiler, G.J. (1998). Seed maturity, storage time and temperature, and media treat-ment effects on germination of two wild sunflowers. Agronomy Journal 90:221-226.

Srivastava, A.K. and Dey, S.C. (1982). Physiology of seed dormancy in sunflower.Acta Agronomica Academiae Scientarum Hungaricae 31: 70-80.

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Steinbach, H.S., Benech-Arnold, R.L., Kristof, G., Sánchez, R.A., and MarcucciPoltri, S. (1995). Physiological basis of pre-harvest sprouting resistance in Sor-ghum bicolor (L.) Moench. ABA levels and sensitivity in developing embryos ofsprouting resistant and susceptible varieties. Journal of Experimental Botany 45:701-709.

Steinbach, H.S., Benech-Arnold, R.L., and Sánchez, R.A. (1997). Hormonal regula-tion of dormancy in developing Sorghum seeds. Plant Physiology 113: 149-154.

Takeda, K. (1996). Varietal variation and inheritance of seed dormancy in barley. InNoda, K. and Mares, D. (Eds.), Pre-Harvest Sprouting in Cereals 1995 (pp. 205-212). Osaka, Japan: Center for Academic Societies.

Ullrich, S.E., Han, F., Blake, T.K., Oberthur, L.E., Dyer, W.E., and Clancy, J.A.(1996). Seed dormancy in barley: Genetic resolution and relationship to othertraits. In Noda, K. and Mares, D. (Eds.), Pre-Harvest Sprouting in Cereals 1995(pp. 157-163). Osaka, Japan: Center for Academic Societies.

Ullrich, S.E., Hayes, P.M., Dyer, W.E., Blake, T.K., and Clancy, J.A. (1993). Quan-titative trait locus analysis of seed dormancy in ‘Steptoe’ barley. In Walker-Simmons, M.K. and Ried, J.L. (Eds.), Pre-harvest sprouting in cereals 1992(pp. 136-145). St. Paul, MN: American Society of Cereal Chemistry.

Van Beckum, J.M.M., Libbenga, K.R., and Wang, M. (1993). Abscisic acid andgibberellic acid-regulated responses of embryos and aleurone layers isolatedfrom dormant and nondormant barley grains. Physiologia Plantarum 89: 483-489.

Vegis, A. (1964). Dormancy in higher plants. Annual Review of Plant Physiology15: 185-224.

Visser K., Visser, A.P.A., Cagirgan, M.A., Kijne, J.W., and Wang, M. (1996).Rapid germination of a barley mutant is correlated with a rapid turnover ofabscisic acid outside the embryo. Plant Physiology 111: 1127-1133.

Walker-Simmons, M.K. (1987). ABA levels and sensitivity in developing wheatembryos of sprouting resistant and susceptible cultivars. Plant Physiology 84:61-66.

Walker-Simmons, M.K. (1988). Enhancement of ABA responsiveness in wheatembryos by high temperature. Plant, Cell and Environment 11: 769-775.

Walker-Simmons, M.K. and Sesing, J. (1990). Temperature effects on embryonicabscisic acid levels during development of wheat grain dormancy. Journal ofPlant Growth Regulation 9: 51-56.

Wang, M., Bakhuizen, R., Heimovaara-Dijkstra, S., Zeijl, M.J., De Vries, M.A.,Van Beckum, J.M., and Sinjorgo, K.M.C. (1994). The role of ABA and GA inbarley grain dormancy: A comparative study between embryo dormancy andaleurone dormancy. Russian Journal of Plant Physiology 41: 577-584.

Wang, M., Heimovaara-Dijkstra, S., and Van Duijn, B. (1995). Modulation of ger-mination of embryos isolated from dormant and nondormant grains by manipu-lation of endogenous abscisic acid. Planta 195: 586-592.

Wang, M., van der Meulen, R.M., Visser, K., Van Schaik, H.-P., Van Duijn, B., andde Boer, A.H. (1998). Effects of dormancy-breaking chemicals on ABA levels inbarley grain embryos. Seed Science Research 8: 129-137.

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Wulff, R.D. (1995). Environmental maternal effects on seed quality and germina-tion. In Kigel, J. and Galili, G. (Eds.), Seed Development and Germination(pp. 491-505). New York: Marcel Dekker, Inc.

Xu, N., Coulter, K.M., and Bewley, J.D. (1991). Abscisic acid and osmoticum pre-vent germination of developing alfalfa embryos, but only osmoticum maintainsthe synthesis of developmental proteins. Planta 182: 382-390.

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Chapter 6

Preharvest Sprouting of CerealsPreharvest Sprouting of Cereals

Gary M. PaulsenAndrew S. Auld

INTRODUCTION

Preharvest sprouting of cereals is defined as germination of physiologi-cally ripe kernels before harvest (Derera, 1989b). This simple definition en-compasses numerous factors: maturation, ripening, and after-ripening ofgrain; innate dormancy; the presence of conditions to initiate germination;induction of enzymatic activities; involvement of plant hormones; and suit-ability of the grain for its intended use. The classical definition of germina-tion as the sum total of processes preceding and including protrusion of theradicle/coleorhiza through the surrounding structures (Hilhorst and Toorop,1997) may not be entirely appropriate to the study of preharvest sprouting.Changes that occur early in the endosperm before new seedling tissues maybe so deleterious as to make sprouted grain unfit for many purposes.

Preharvest sprouting is usually associated with prolonged or repeatedrain, heavy dew, high humidity, and low temperature following ripening ofthe grain (Nielsen et al., 1984). The conditions that favor sprouting oftencompound the problem by delaying harvest. Such conditions occur through-out the world: northern and western Europe; parts of Africa; tropical andsemitropical Asia, including southeastern China; northern Australia; north-ern and northwestern areas of the United States and adjacent areas in Can-ada; and a broad band across South America (Derera, 1989). The problemmay be exacerbated by cultivation of susceptible crops in those areas. Anexample is production of white wheat (Triticum aestivum L.), which has lit-tle resistance to preharvest sprouting in the northern wheatbelt of Australia.However, damage also occurs with some frequency even in areas whereconditions do not normally favor sprouting. For instance, in Kansas, themajor wheat state in the United States, hot, dry weather following ripening

This chapter is contribution number 02-316-B from the Kansas Agricultural Experi-ment Station.

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of resistant hard red wheat usually results in little preharvest sprouting.Still, significant damage occurred in parts of the state during 1979, 1989,1993, and 1999, when conditions were particularly favorable.

Instances of preharvest sprouting have been reported for all cereals.Most damage occurs to common wheat because it is the most widely grownof all cereals, including cultivation in areas where sprouting is likely to oc-cur. Many cultivars are susceptible, and sprouting is highly deleterious tosome of the products.

Rye (Secale cereale L.) is particularly susceptible to preharvest sprout-ing because the flowers are cross-pollinated, and the open structures of theglumes allow water to reach the grain (Derera, 1989b). Preharvest sproutingof barley (Hordeum vulgare L.) damages the quality of the grain for bakingand viability of the kernels for malting. However, germination may increasedigestibility of both crops and enhance their feeding value for livestock.Sprouting of oat (Avena sativa L.) occurs episodically in some areas, partic-ularly northern regions, but has little effect on the quality of the grain forfeed. Triticale (×Triticosecale Wittmack), like rye, is extremely liable topreharvest sprouting. Sprouting has the same deleterious effect on bakingquality of triticale as on wheat, but the bulk of the crop is used for livestockand its value is affected little. Preharvest sprouting of maize (Zea mays L.)is usually associated with vivipary (Smith and Fong, 1993) because thegrain is protected by the husk from the moist conditions that promote ger-mination in other cereals. Sorghum [Sorghum bicolor (L.) Moench] andpearl millet [Pennisitum glaucum (L.) R. Br.] are rarely subject to preharvestsprouting because of the semiarid nature of the regions where they aregrown. However, grain of both species sprouts when conditions are appro-priate. Japonica rice (Oryza sativa L.), which is usually grown in more tem-perate regions, sprouts more easily than Indica rice of the tropics, which ishighly resistant (Yamaguchi et al., 1998).

The most extensive survey of direct losses to producers from preharvestsprouting of cereals was reported by Derera (1989c). Average annual lossesin 37 countries totaled over US$450 million, mostly to wheat, from 1978 to1988. However, the major cereal-producing countries of China, India,USSR, and Argentina were not included in the survey, and estimates werenot available from the United States and several other countries. Sproutingof durum (Triticum durum Desf.) wheat in the northern United States alonecaused several hundred million dollars of damage over a decade (Dick et al.,1989). It is likely that total worldwide direct annual losses currently ap-proach US$1 billion.

Direct economic losses to producers from preharvest sprouting occur inseveral ways. The yield may be reduced by loss of dry matter and shatteringof the grain, the volume density (test weight) may decrease from loss of dry

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matter and irreversible swelling of the kernels, and suitability of the grainfor many food products may be diminished. Because payments to producersin the United States and many other countries are determined by the yield,volume density, and grade of the grain, any of the effects of sprouting re-duce their income. In the United States, for instance, more than 4 percentdamaged kernels—including sprouted kernels—causes hard wheat to berated Grade 3 or lower and unacceptable for bread making. The loss in valuefrom diminished quality, however, is usually offset by use of sprouted grainfor livestock feed.

Indirect losses should be added to the total economic losses from pre-harvest sprouting of cereals. Traditional markets are lost when exporterscannot supply sound grain to customers (Briggle, 1979). Producers in partsof China would benefit from growing white wheat because of higher pay-ments from the government but must grow red wheat because of the hazardof sprouting damage (Paulsen, 1985). Production of some cereals in the hu-mid tropics is limited, in part, by the possibility of preharvest sprouting.

THE PREHARVEST SPROUTING PROCESS

Absorption of moisture by kernels is influenced by morphology of theinflorescence, characteristics of the seedcoat, turgor of the embryo, andchemical properties of the caryopsis (King, 1989). Environmental factors,particularly temperature, also affect imbibition by influencing the proper-ties of water (Murphy and Noland, 1982). Dry grain (9 to 12 percent mois-ture) has an extremely low water potential, –400 MPa in the case of wheat,and so readily imbibes water (Shakeywich, 1973). Fifty percent germina-tion occurs at a threshold water potential of 0.8 to 1.0 MPa or about 45 per-cent seed moisture content (King, 1989). Cereals do not have impermeableseedcoats as do legumes, and the critical moisture content for germinationin freely available water is reached in about 3 h. However, cultivars differsubstantially in the rate of imbibition. Many factors have been implicated incontrolling imbibition, but no single factor has been identified. Conditionsthat influence imbibition by wheat and other cereals were reviewed by King(1989). Imbibition is increased by features associated with awns in wheatand is affected by waxiness, pubescence, and angle of the inflorescence inbarley. Grain hardness, color, restriction by the seedcoat, thickness of thetesta and other layers, size, and surface:volume ratio are implicated in somestudies but not others (King, 1989). The rate of drying of the spike and grainafter moisture becomes unavailable is also likely to affect sprouting, but ap-pears to be determined solely by evaporation and does not differ amongcultivars. Temperature affects imbibition by influencing the viscosity of

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water and probably the wetability of tissues (Vertucci and Leopold, 1986),as well as the rate of drying by evaporation.

Water enters the grain most rapidly via tissues that overlay the embryo(Evers, 1989). King (1989) concluded that the main path of water to the em-bryo must be laterally through the pericarp. Starch in the endosperm of ce-reals is much more hydrophobic than the contents of the embryo (Chungand Pfost, 1967).

Movement of the water front through the kernel initiates numerous pro-cesses in the embryo, endosperm, and associated tissues. Absorbed gassesare released, membranes are reorganized, mitochondria develop, endoge-nous enzymes are activated, and new enzymes appear by de novo synthesis(McDonald, 1994). Most of the deleterious changes during sprouting occurfrom mobilization of reserves in the endosperm. Most attention has beengiven to hydrolysis of starch, but many other substrates in the endosperm—proteins, lipids, phytin, etc.—are degraded to provide substance for the em-bryo and developing seedling. Although the changes in the endosperm aremost prominent, they are mostly controlled by the embryo/scutellum (King,1989).

Investigations of biochemical and physiological changes in cereals dur-ing preharvest sprouting have emphasized the enzymes involved. Enzymescatalyze the biochemical processes, and changes in their activities areamong the most pronounced effects of preharvest sprouting. They are alsoresponsible for most of the deleterious changes that occur. Several mea-sures of preharvest sprouting are based on changes in enzyme activity.

Hydrolysis of starch in the endosperm to simple sugars for use by the em-bryo involves numerous enzymes: endoamylases ( -amylases), debranchingenzymes (R-enzyme, pullulanase), isoamylase, exoamylase ( -amylase),and -glucosidase (maltase) (Beck and Ziegler, 1989). -Amylase is theonly enzyme that can hydrolyze raw starch. It cleaves 2(164) glucosidicbonds in amylose and amylopectin and is often considered to be the key tothe problem of preharvest sprouting (Duffus, 1989).

-Amylase in cereals is commonly divided into two types, an endoge-nous late maturity, green, or low-pI group and a germination or high-pIgroup that is associated with sprouting (Kruger, 1989). Numerous isozymesoccur within both groups, and differences between groups are not distinct.Late-maturity -amylase also occurs during germination, and some iso-zymes that form during maturation have pIs that are typical of isozymes thatappear during germination (Mares and Mrva, 1993). Production of late-matu-rity -amylase is controlled by the pericarp or embryo, whereas -amylasesthat form during germination are associated with the aleurone and/or thescutellum (Kruger, 1989).

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The relative importance of -amylase activity that was retained in thepericarp or formed during late maturity, during germination before matura-tion, or during germination after maturation of wheat was assessed by Lunnand colleagues (2001). Late-maturity -amylase was most widespread, oc-curring in 25 of 32 cultivar × location × year instances where sprouting wasidentified. However, -amylase that formed during sprouting occurred in21 of the 32 instances and was primarily responsible for damage to the grainfrom preharvest sprouting. Late-maturity, low-pI -amylase in the wheatcultivar Chinese Spring was controlled by a single recessive gene on thelong arm of chromosome 6BL, and synthesis of high-pI -amylase in thealeurone also involved a gene on the long arm of chromosome 6BL (Mrvaand Mares, 1998).

Debranching enzymes, including isoamylase, as their name suggests,hydrolyze -(1 6)-glucosidic bonds in amylopectin to accelerate break-down of the starch by -amylase (Kruger, 1989). The enzyme is formed dur-ing early stages of maturation, and at least two isozymes occur in wheat. Insome species, the enzyme accumulates in an inactive form that is liberatedby proteolysis during germination (Beck and Ziegler, 1989).

-Amylases hydrolyze alternate (164) bonds of starch to form maltose.They develop during maturation and occur in free and bound forms in ripegrain (Beck and Ziegler, 1989). The enzyme is present in both the pericarpand the endosperm, although the former disappears during maturation andonly the latter structure contains the enzyme when the grain ripens (Kruger,1989). -Amylases may be important in preharvest sprouting of wheat ifinsufficient activity relative to -amylases leads to an accumulation ofdextrins that make bread crumbs sticky (Duffus, 1989).

Maltase is generally present at low levels in mature grain and increasesby de novo synthesis during germination. It is mostly produced in thescutellum and secreted into the endosperm (Beck and Ziegler, 1989).

Proteolytic enzymes function during sprouting to mobilize N for the em-bryo and seedling and to release bound or inactive enzymes. -Amylase,debranching enzymes, and probably other enzymes are complexed withproteins during maturation and then freed by proteolysis during germina-tion (Beck and Ziegler, 1989).

Many types of proteolytic enzymes—endopeptidases, carboxypeptidas-es, aminopeptidases, etc.—are associated with sprouting. Knowledge oftheir roles is complicated by their complexity, difficulty of extraction andpurification, and differing reactions to assay conditions and substrates(Kruger, 1989). An endopeptidase that attacks modified gluten occurs innongerminated wheat, and it and another endopeptidase increase rapidlyduring germination (McMaster et al., 1989). The first enzyme was appar-ently distributed throughout the endosperm, whereas the enzyme that was

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activated by germination was derived from the aleurone and scutellum. Ac-tivity of the enzymes in damaged grain affected quality of the dough forbread, and activity during processing affected the quality for alkaline noo-dles. Nongerminated barley, in contrast, contained endopeptidase that hy-drolyzed edistin but not gelatin or hordein (Jones and Wrobel, 1993). Ger-mination induced a number of proteinases, most of which were associatedwith aleurone, scutellar, and endosperm tissues.

A carboxypeptidase in the endosperm of wheat increases throughoutmaturation, whereas one in the outer layers of the kernel disappears (Kruger,1989). During germination, activity of the enzyme in the endosperm nearthe scutellar epithelium increases, apparently due to dissipation of inhibi-tors.

Lipases have little or no activity in nongerminated cereals (Jensen andHeltved, 1982). During germination, activity appears first in the scutellum,followed by the scutellum-endosperm interface, and then gradually pro-gresses throughout the endosperm. Enhanced activity of lipases may affectthe viability of sprouted kernels during storage (Kruger, 1989). However,the increase in lipases is usually much smaller than the change in -amylase(Fretzdorff, 1993).

Activity of many other enzymes increases markedly during sprouting ofcereals. Phytases increase to release phosphorus for the new seedling.Monophenol oxidase and polyphenol oxidase may increase up to 33-foldand cause the gray crumb discoloration of bread and the off color of noodlesmade from sprouted wheat (Kruger, 1989). However, the increase in poly-phenol oxidase is typically much less than -amylase, and little of the en-zyme occurs in flour after milling (Kruger and Hatcher, 1993). Catalasesand peroxidases catalyze oxidative reactions that may affect the rheologicalproperties of dough. Other enzymes that increase during sprouting, such asribonucleases, are important for the developing seedling but have no knowneffects on cereal products.

PHYSIOLOGICAL CONTROLOF PREHARVEST SPROUTING

Preharvest sprouting is affected at numerous levels by factors rangingfrom inhibitors in awns (bracts) to control of -amylase synthesis bygibberellic acid (GA) (Gale, 1989). As discussed in Chapter 5, many ofthese factors involve dormancy, which is the inability of a viable, matureseed to germinate even under favorable conditions (Hilhorst and Toorop,1997). Of the two types of dormancy— coat-imposed dormancy derivedfrom the presence of endosperm plus pericarp (plus glumellae in the case of

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barley) and true embryo dormancy—only the former occurs in cereals. Im-mature embryos of barley, rice, and wheat, for example, rapidly germinatewhen they are removed from developing kernels and placed in water orother media (Kermode, 1990).

All of the factors discussed earlier that affect imbibition also influencesprouting. Resistance to expansion of the germinating kernel and its em-bryo by the pericarp and testa may also inhibit germination (Wellington,1956). Other unknown factors may cause differences in the rate of germina-tion among genotypes even when the rate of imbibition and other traits aresimilar (Gale, 1989).

Inhibitors of various types play central roles in preharvest sprouting. Theglumes (bracts) of wheat, for instance, contain an unknown inhibitor thatdelays sprouting and is simply inherited (Derera and Bhatt, 1980; Wu andCarver, 1999). Similarly, the well-known resistance to preharvest sproutingof red wheats relative to white wheats has been attributed to precursors ofthe pigment phlobaphene in the testa layer of the former (Miyamoto,Tolbert, and Everson, 1961). These compounds, catechin and tanninlikematerials, occurred in lower amounts in white wheats than in red wheats, inwhich they declined during after-ripening to permit germination. Pigmentsin the seedcoat may be part of a two-factor system that inhibits germinationdirectly or by interfering with gaseous exchange (Mares, 1998).

Numerous plant growth substances are directly implicated in preharvestsprouting. In addition to the pregerminative action of gibberellic acid dis-cussed in Chapter 5, the postgerminative role of GA from the embryo andscutellum in inducing synthesis of -amylase in the aleurone is well known(Beck and Ziegler, 1989). Debranching enzymes, maltase, some protein-ases, phytase, ribonuclease, and others are also induced by GA, but themechanisms may differ. Whereas de novo synthesis and secretion of -amylase are stimulated by GA, other enzymes may be activated by GA orwould increase even without GA (King, 1989). In barley and presumablyother cereals, GA promotes accumulation of a-amylase mRNA in thealeurone and involves synthesis of a protein factor for efficient expression(Muthukrishnan, Chaudra, and Maxwell, 1983). The GA acts as a positiveregulator of expression of -amylase genes in vivo in barley (Chandler andMosleth, 1989).

Abscisic acid has many imputed functions in addition to regulating de-velopmental changes from maturation to germination (Kermode, 1990).Dormancy of cereals is roughly proportional to their abscisic acid (ABA)content, suggesting that the growth substance is involved in both the initia-tion and maintenance of nongerminability (King, 1989). Other work sug-gests that wheat cultivars that differ in dormancy have similar contents ofABA but vary in sensitivity to the compound (Walker-Simmons, 1987). The

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role of this hormone in controlling the timing of dormancy release in cerealgrains is thoroughly treated in Chapter 5. In addition, and from a post-germinative standpoint, it has been found that an ABA-responsive proteinkinase mRNA mediated the suppression of GA-inducible genes in thealeurone of wheat (Walker-Simmons et al., 1998).

Other growth substances—jasmonic acid, ethylene, and cytokinins—modify germination of many species but have not been studied extensivelyin cereals (Hilhorst and Toorop, 1997). Liu and colleagues (1998) con-cluded that indole-3-acetic acid (IAA) inhibited germination and acted inconcert with GA and cytokinins to regulate the process. The observationthat tryptophan, a purported precursor of IAA, inhibited sprouting of resis-tant wheat cultivars supports a role for the auxin in controlling germination(Morris et al., 1988).

Several proteins, mostly albumins, that inhibit endogenous -amylaseoccur in wheat and barley (Gale, 1989). The proteins increase during ger-mination and may control -amylase activity. Only proteins from sprout-ing-resistant genotypes inhibited -amylase from a sprouting-susceptiblegenotype of wheat (Abdul-Hussain and Paulsen, 1989). However, addingethylenediaminetetraacetic acid (EDTA) to chelate calcium caused inhibi-tion by all genotypes, suggesting that the proteins interacted with the metal.Phytic acid from the bran also inhibited -amylase activity in wheat, againby lowering the level of the calcium cofactor (Cawley and Mitchell, 1968).

QUALITY OF PRODUCTS FROM SPROUTED CEREALS

The consequences of preharvest sprouting directly depend on the typesof products for which the cereal is intended and on the processing methodsused. Severely sprouted grain might be blended with sound grain in somecases and almost always has considerable residual value as livestock feed.Sprouting might even increase the value of cereals for feed by making themmore palatable and digestible.

Breads

Breads baked from hard wheats are affected more than most products bypreharvest sprouting of the grain. Production of the bread is complicated byextreme stickiness of the dough, which necessitates special handling insmall bakeries and can disrupt operations of large bakeries. Even slicingbread made from sprouted wheat can be difficult, and the resulting loavesare often cavitated and grayish.

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Extensive studies with field-sprouted wheats by Kulp, Roewe-Smith,and Lorenz (1983) and Lorenz and colleagues (1983) illustrate the problem.Sprouting weakened the dough strength, decreased the amylograph peakviscosities, and caused poor handling and machining properties. Loaf vol-ume increased, but the internal quality was poor. Thickening ability ofstarch from sprouted wheat was adversely affected.

Stickiness of dough is usually attributed to extensive enzymic hydrolysisof damaged starch and altered rheological properties to proteolytic en-zymes (Kruger, 1989). Kulp, Roewe-Smith, and Lorenz (1983) and Lorenzand colleagues (1983) also concluded that elevated -amylase and pro-teinase activities were responsible. However, electron microscopy and X-raydiffraction found no changes in starch that were attributable to sprouting.Sticky dough might also be caused by the limit dextrins that result from ex-cessive -amylase relative to -amylase activity and possibly -amylase todebranching activity (Duffus, 1989). Proteinases and lipases that open thestarch granule to amylosis might also be involved.

Hearth breads appear to be degraded less than Western-style pan breadsby sprouted grain. Seven of nine international leavened and unleavenedbreads from flour from soft white wheat that sprouted in the field had ac-ceptable quality (Finney et al., 1980).

Cakes and Cookies

Field sprouting of soft wheats had little effect on crumb properties ofsponge cake but increased the cake volume at low levels and decreased it athigh levels of sprouting (Finney et al., 1981). Sprouting of hard wheats alsoincreased the volume and coarsened the grain of yellow cake (Lorenz et al.,1983); however, the cake texture was smoother and softer. In other studies,sprouting caused poor baking quality, a depressed center, coarse grain, anda firm texture in cakes (Lorenz and Valvano, 1981). When flour fromsprouted grain was used for cookies, the spread increased and the top grainscore improved, but the crust darkened.

Speciality Batters

High -amylase in sprouted wheat generally reduces the quality of bat-ters for many uses (Nagao, 1995). Batter for coating fish and vegetables astempura loses its light and viscous character and coats poorly. Batter fortakoyaki, coated octopus tentacles, loses its shape. When used for Japanesemuffins with a sweet bean filling, the batter may not be viscous enough tocover the contents.

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Pasta

Sprouting affects both the processing and quality of the many kinds ofnoodles that are made from wheat. In dry noodles, high -amylase weakensthe dough so that the noodles cannot support their own weight and breakduring the dehydration process (Nagao, 1995). For wet noodles, wherecolor, brightness, and texture are of major importance, the enzymes that in-crease during sprouting seriously affect product quality (Kruger, Hatcher,and Dexter, 1995). Cantonese noodles were slightly less bright when theywere prepared from sprouted wheat but had similar textural properties asnoodles from sound wheat. Raw noodles differed only slightly in firmnessand resistance to compressibility.

Changes in noodle quality are usually attributed to -amylase, pro-teinase, and polyphenol oxidase enzymes that increase during sprouting(Kruger, 1989). -Amylase might be most problematic, since it typicallyincreases several-thousand-fold during sprouting, and over 75 percent ofthe activity in whole meal occurs in the flour (Kruger and Hatcher, 1993).Effects of increased proteinase activity may be overshadowed by the -am-ylase (Kruger, 1989). Polyphenol oxidase, in contrast to -amylase, in-creases only about 2.5-fold during sprouting and is localized in the bran sothat only about 1 percent of the activity occurs in the flour. Low water ab-sorption of flours may limit the mobility between the enzymes and theirsubstrates, and the brief processing time may limit the period for deteriora-tion to occur during preparation of noodles compared with bread (Kruger,Hatcher, and Dexter, 1995).

Alcoholic Beverages and Glucose Syrups

Amylosis is a primary step in the processing of cereals for beverages andsyrups. Starch is hydrolyzed to dextrins for beer and glucose syrup but mustbe completely converted to fermentable sugars for production of alcoholand spirits (Peiper, 1998). Malt from barley is generally used for controlled,uniform amylosis in most fermentation processes. Cultivars that have lowor no dormancy are greatly preferred for malting (Aastrup, Riis, andMunck, 1989). However, preharvest sprouting of the barley may shorten itsviability during storage, lower conversion of the malt and extractability offermentable material, and increase the growth of molds (Kruger, 1989).

Seed Quality

The quality of sprouted grain for seed concerns seedsmen and farmers.Wheat seed that is severely sprouted loses viability rapidly, is easily dam-

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aged, and deteriorates quickly during storage (Elias and Copeland, 1991;Barnard and Purchase, 1998). The emergence percentage in the field maybe substantially lower than the germination percentage in the laboratory,and stands may be reduced further by treatment of the seed with fungicides(Barnard and Purchase, 1998). Adverse storage conditions may acceleratethe decline in seed germination and seedling vigor (Stahl and Steiner,1998). However, it is doubtful that severely sprouted seed appears often incommercial channels.

Wheat seed that has low or incipient levels of sprouting (mean Hagbergfalling number of 107) but is otherwise sound may be used for planting(Foster, Burchett, and Paulsen, 1998). Germination and emergence fromdeep planting and field establishment declined in some cultivars, but grainyields were not affected even after storing the seed for 27 months.

MEASUREMENT OF PREHARVEST SPROUTING

Evaluation of damage to grain from preharvest sprouting and of resis-tance to the problem involves numerous considerations (Mares, 1989; Wuand Carver, 1999). Routine assays of sprouting mostly involve proper stor-age and preparation of the samples and measurement of sprouting damage.Experimental evaluation of sprouting resistance has similar requirementsplus the inclusion of a suitable wetting treatment to induce sprouting.

Sampling

Grain should be sampled at uniform stages of development to avoid dif-ferences in dormancy during maturation. This might be at physiologicalmaturity, when the grain contains 25 to 35 percent moisture, or at harvestripeness, when the grain moisture is at 12 to 13 percent. Samples may be ei-ther assayed immediately or dried to 15 percent or less moisture and storedfor future use. Drying with ambient air is often satisfactory and forced air, ifused, should not exceed 30°C and only for the briefest duration. Lyophili-zation is not recommended in most cases because of the possibility of freezedamage to the embryo and other parts of the grain. Once the sample is dry, itcan be held at room temperature, where it will after-ripen naturally but incurlittle change in -amylase activity and most constituents. Alternatively,dried samples may be held at –20°C to arrest the after-ripening process andpreserve dormancy. If the intact spike is used, 5 to 10 cm of culm should beleft for handling and if the grain is threshed, hand rubbing or dissectionshould be used instead of mechanical methods to avoid damage to the ker-

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nels. Detailed discussions of these considerations are given by Mares(1989).

Controlled Sprouting

Assaying sprouting in intact spikes is often preferred over other methodsbecause it incorporates differences in wetting, water movement, inhibitorsin the glumes, and other factors. Rain simulators of various types, mistingchambers, immersion in water, burial in moist sand, and other methods areused. Rain simulators do not duplicate the kinetic energy, velocity, and ran-dom size of natural rain (King, 1989), but consistent results among variousmethods suggest that the effect is small (Mares, 1989). Sprouting of kernelsin the spike can be assessed visually, by dissecting the grain, and by analyz-ing -amylase activity.

Sprouting of kernels on commercial germination paper, filter paper, orother media, often in petri dishes, is practiced routinely. The technique iscriticized for its lack of physiological integrity, particularly in water con-tent, but it does measure relative dormancy under standard conditions(Mares, 1989). Results may be expressed as percentage of kernels germi-nating, time to 50 percent germination, or other expressions. A germinationpromptness index,

GPI =d

ii

i

n

,1

(6.1)

where di = number of kernels germinating on day i, incorporates both therate and magnitude of germination.

Excised embryos are germinated on media with various adjuncts be-cause of the importance of their response to preharvest sprouting. Sensitiv-ity of the embryos to ABA, catechins, or other substances is associated withresistance to sprouting and controlled simply by a few genes in wheat andbarley (Gale, 1989). Embryos are easily dissected from kernels and germi-nate rapidly in water or basal media.

Measuring Sprouting

Various methods are available for measuring sprouting, and their choicedepends on the requirements for the test. Visual counting of sprouted ker-nels and the Hagberg falling number are generally used for commercialsamples, whereas these and other methods, many of which measure activi-ties of enzymes, are used experimentally.

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Visual counting of sprouted kernels is usually the first measure of dam-age to grain at local elevators and terminal elevators. In the United States,sprouted wheat is rated by the Federal Grain Inspection Service (1997) as“kernels with the germ end broken open from germination and showingsprouts or from which the sprouts have broken off” (p. 32). The method,which judges kernels to be sprouted or not sprouted, lacks precision andtypically has extremely high coefficients of variation for experimental use.In addition, considerable damage from elevated -amylase activity may oc-cur well before any seedling structures are evident.

The Hagberg falling number method (Falling Number Corp., Huddinge,Sweden) measures the time in seconds for a plunger to fall through thegelatinized starch in a slurry of ground grain. Values range from 60 forhighly sprouted grain to 500 or higher for sound grain; a minimum of 250 to300 is generally required for wheat grain for bread. The procedure is af-fected by a number of factors but is rapid, simple, and gives good precision.It has been adopted as the official method by several associations and muchof the grain industry. The relatively large sample sizes, 300 g for the sampleand 7 g for the assay, are barriers for many experimental uses.

The stirring number as measured by the Rapid Visco Analyzer (FossTechnology Corp., Eden Prairie, Minnesota) is another viscometric methodthat uses a slightly smaller sample (4 g) than the falling number procedureand is highly reproducible. The Brabender amylograph (C.W. BrabenderCo., South Hackensack, New Jersey) measures changes in the viscosity of aflour-water paste with increased temperature. The method detects low lev-els of sprouting and other properties. It has the disadvantages of requiring alarge sample (60 g) and long running time (ca. 60 min).

Falling number, stirring number, and amylograph peak viscosity valuesare affected by sample size, temperature, and often by barometric pressure(Koeltzow and Johnson, 1993). The values are usually highly correlated,but results cannot be easily converted from one method to another.

A number of methods are available for measuring -amylase. Oldermethods that determine gas production or reducing power are used infre-quently because they are time-consuming and often require specializedequipment. Several procedures measure -amylase activity with a dye-labeled starch substrate. The action of the enzyme on the substrate liberatesthe soluble dye, which is usually measured spectrophotometrically. Themethod requires a constant temperature water bath, shaker, centrifuge,spectrophotometer, and other equipment and is not suited for many uses.However, it provides a direct measure of -amylase activity, which is oftenof interest for experimental purposes.

Nephelometry, which measures scattering of light by suspended parti-cles, also directly measures -amylase activity. As the -limit dextrin sub-

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strate is hydrolyzed by the enzyme, the nephelor decreases linearly. Themethod is highly sensitive to low levels of -amylase but requires substan-tial expertise for its use (Kruger and Hatcher, 1993). A kinetic microplatemodification of the method has been described (Kruger and Hatcher, 1993).

Several procedures measure -amylase that diffuses from sectionedgrain into an agar-starch substrate. Diffusion of the enzyme is deleted withiodine-iodide or by using a dye-labeled starch; the logarithm of activity ismeasured by the diameter of the digested area. The procedure takes aboutone day to complete but requires little equipment and expense.

Other enzymes, including proteinases, oxalate oxidase, and lipases, havebeen suggested as measures of preharvest sprouting. Proteinases offer littleadvantage over -amylase, which causes most of the damage during sprout-ing and can be assayed by a variety of methods. Oxalate oxidase might beuseful for detecting early sprouting, but it may not be applicable to severelysprouted samples (Fretzdorff and Betsche, 1998). The increase in lipase ac-tivity during sprouting can be visualized by hydrolysis of nonfluorescentfluorescein dibutyrate to fluorescent fluorescein (Jensen and Heltved,1982).

Monoclonal antibodies are also used to measure sprouting by detectingspecific enzymes or other constituents. The antibodies are typically labeledwith fluorescein to visualize the reaction. Several commercial systems thatemploy the method for routine sampling have been developed, and the pro-cedure is extremely useful for determining specific changes during sprout-ing.

CONTROLLING SPROUTING BY BREEDING

Similar to many other plant adversities, preharvest sprouting is con-trolled most effectively and economically by genetic resistance. However,as noted by Derera (1989b), breeding practices by scientists and productionpractices by farmers often work against dormancy of cereals. Breeders, forinstance, often shuttle their experimental lines between the field and thegreenhouse or between summer and winter nurseries to raise as many gen-erations as possible each year. Farmers in northern latitudes may plant win-ter cereals from seed that was recently harvested. In some cases, industrymay require cereals that have little or no dormancy, as with barley for malt-ing (Aastrup, Riis, and Munck, 1989).

Breeding for resistance to preharvest sprouting can take many approachesbecause of the numerous morphological, physiological, and biochemicalfactors that influence the trait. Multiple sources of genetic resistance are

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available in wheat, barley, and rye (Derera, 1989a), and their dissimilar ge-netic mechanisms suggest that different genes can be “pyramided” into sin-gle genotypes to create highly resistant genotypes (Allan, 1993). Geneticengineering may likewise hold considerable promise for increasing resis-tance of cereals to preharvest sprouting (Anderson, Sorrels, and Tanksley,1993).

Resistance to preharvest sprouting is strongly associated with red graincolor in wheat, and much of the effort to improve the trait has focused onwhite wheat (Gale, 1989). Most studies find that resistance of white wheatto preharvest sprouting is a quantitative trait (e.g., Upadhyay and Paulsen,1988; Paterson and Sorrells, 1990; Allan, 1993). This result may be associ-ated with the different dormancy mechanisms that occur (Paterson and Sor-rells, 1990). Other studies indicate that dormancy is controlled by one ortwo recessive genes (Bhatt, Ellison, and Mares, 1983).

Four quantitative trait loci (QTLs) for dormancy were detected in barley(Han et al., 1996), and five QTLs for dormancy were detected in rice (Lin,Sasaki, and Yano, 1998). Wheat had three QTLs that explained more than80 percent of the total phenotypic variance in seed dormancy (Kato et al.,2001). A major QTL was located on the long arm of chromosome 4A, andtwo minor QTLs were on chromosomes 4B and 4D. Comparative maps sug-gested a homologous relationship between the major QTL and barley geneSD4.

‘Clark’s Cream’ white wheat illustrates the use of resistance in an un-adapted cultivar to improve resistance to preharvest sprouting in a moderncultivar. One of the present authors (GMP) received a bushel of ‘Clark’sCream’ seed from Mr. Earl Clark, a breeder-farmer who developed manyimportant early cultivars for the Great Plains, for agronomic studies in1976. The cultivar had a low level of sprouting after persistent rains in 1979,and subsequent studies showed that it expressed both high dormancy andlow -amylase production (McCrate et al., 1981). The traits were later asso-ciated with extreme embryo sensitivity to an endogenous inhibitor (Morriset al., 1989). An earlier report by Heyne (1956) stated that Clark normallyleft his experimental lines in the field for eight weeks after they ripened,suggesting that modifiers accumulated for resistance to sprouting. Broad-sense heritability estimates for sprouting resistance were moderate, and thetrait was quantitatively inherited (Upadhyay and Paulsen, 1988). ‘Clark’sCream’ was used to develop the sprouting-resistant cultivar Cayuga andidentify four genetic markers for the trait (Anderson, Sorrels, and Tanksley,1993).

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CONTROLLING SPROUTING IN THE FIELD

Few remedies are available for preventing preharvest sprouting whenweather conditions promote germination. Planting of resistant species andcultivars, if they are available, is obvious. In some cases, cultivars that havethe appropriate maturity to ripen before or after seasonal rains that causesprouting may be selected.

Prompt harvest of the grain is usually the best means to prevent pre-harvest sprouting. In some cases, this involves harvesting immediately afterthe grain ripens, i.e., contains 12 to 13 percent moisture and can safely bestored. In other cases, the grain must be harvested earlier at a moisture levelof 16 to 20 percent and artificially dried at extra expense. Grain that isswathed before it is ripe may be more dormant and resistant to sproutingthan grain that is allowed to dry while standing. However, grain that isswathed dries slowly, and rain may increase the level of preharvest sprout-ing.

REFERENCES

Aastrup, S., Riis, P., and Munck, L. (1989). Controlled removal of dormancy ren-ders prolonged storage of sprouting resistant malting barley superfluous. InRinglund, K., Mosleth, E., and Mares, D.J. (Eds.), Fifth International Sympo-sium on Preharvest Sprouting in Cereals (pp. 329-337). Boulder, CO: WestviewPress.

Abdul-Hussain, S. and Paulsen, G.M. (1989). Role of proteinaceous -amylase en-zyme inhibitors in preharvest sprouting of wheat grain. Journal of Agricultureand Food Chemistry 37: 295-299.

Allan, R.E. (1993). Genetic expression of grain dormancy in a white-grain wheatcross. In Walker-Simmons, M.K. and Reid, J.L. (Eds.), Pre-Harvest Sproutingin Cereals 1992 (pp. 37-46). St. Paul, MN: American Association of CerealChemists.

Anderson, J.A., Sorrels, M.E., and Tanksley, S.D. (1993). RFLP analysis of genom-ic regions associated with resistance to preharvest sprouting in wheat. Crop Sci-ence 33: 453-459.

Barnard, A. and Purchase, J. (1998). The effect of seed treatment and preharvestsprouted seed on the emergence and yield of winter wheat in South Africa. InWeipert, D. (Ed.), Eighth International Symposium on Pre-Harvest Sprouting inCereals (pp. 26-35). Detmold, Germany: Association of Cereal Research.

Beck, E. and Ziegler, P. (1989). Biosynthesis and degradation of starch in higherplants. Annual Reviews of Plant Physiology and Plant Molecular Biology 40:95-117.

Bhatt, G.M., Ellison, F.W., and Mares, D.J. (1983). Inheritance studies on dor-mancy in three wheat crosses. In Kruger, J.E. and LaBarge, D.E. (Eds.), Third

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International Symposium on Preharvest Sprouting in Cereals (pp. 274-278).Boulder, CO: Westview Press.

Briggle, L.W. (1979). Pre-harvest sprout damage in wheat in the U.S. Cereal Re-search Communications 8: 245-250.

Cawley, R.W. and Mitchell, T.A. (1968). Inhibition of wheat -amylase by branphytic acid. Journal of the Science of Food and Agriculture 19: 106-108.

Chandler, P.M. and Mosleth, E. (1989). Do gibberellins play an in vivo role in con-trolling alpha-amylase gene expression? In Ringlund, K., Mosleth, E., andMares, D.J. (Eds.), Fifth International Symposium on Preharvest Sprouting inCereals (pp. 100-109). Boulder, CO: Westview Press.

Chung, D.S. and Pfost, H.B. (1967). Adsorption and desorption of water vapor bycereal grains and their products. Transactions of the ASAE 10: 549-555.

Derera, N.F. (1989a). Breeding for preharvest sprouting tolerance. In Derera, N.F(Ed.), Preharvest Field Sprouting in Cereals (pp. 111-128). Boca Raton, FL:CRC Press.

Derera, N.F. (1989b). The effects of preharvest rain. In Derera, N.F (Ed.), Pre-harvest Field Sprouting in Cereals (pp. 1-25). Boca Raton, FL: CRC Press.

Derera, N.F. (1989c). A perspective of sprouting research. In Ringlund, K., Mosleth,E., and Mares, D.J. (Eds.), Fifth International Symposium on Preharvest Sprout-ing in Cereals (pp. 3-11). Boulder, CO: Westview Press.

Derera, N.F. and Bhatt, G.M. (1980). Germination inhibition of the bracts in rela-tion to pre-harvest sprouting tolerance in wheat. Cereal Research Communica-tions 8: 199-201.

Dick, J.W., Hansen, D., Holm, Y.F., and Cantrell, R.G. (1989). Pearling and millingof sprouted durum wheat. In Ringlund, K., Mosleth, E., and Mares, D.J. (Eds.),Fifth International Symposium on Preharvest Sprouting in Cereals (pp. 344-350). Boulder, CO: Westview Press.

Duffus, C.M. (1989). Recent advances in the physiology and biochemistry of cerealgrains in relation to pre-harvest sprouting. In Ringlund, K., Mosleth, E., andMares, D.J. (Eds.), Fifth International Symposium on Preharvest Sprouting inCereals (pp. 47-56). Boulder, CO: Westview Press.

Elias, S. and Copeland, L.O. (1991). Effect of preharvest sprouting on germination,storability, and field performance of red and white wheat seed. Journal of SeedTechnology 15: 67-78.

Evers, A.D. (1989). Grain morphology in the sprouting context. In Ringlund, K.,Mosleth, E., and Mares, D.J. (Eds.), Fifth International Symposium on Pre-harvest Sprouting in Cereals (pp. 57-64). Boulder, CO: Westview Press.

Federal Grain Inspection Service (1997). Wheat. In Grain Inspection Handbook,Book II (Chapter 13). Washington, DC: Grain Inspection, Packers and Stock-yards Administration, U.S. Department of Agriculture.

Finney, K.F., Natsuaki, O., Bolte, L.C., Mathewson, P.R., and Pomeranz, Y. (1981).Alpha-amylase in field-sprouted wheats: Its distribution and effect on Japanese-type sponge cake and related physical and chemical tests. Cereal Chemistry 58:355-359.

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Finney, P.L., Morad, M.M., Patel, K., Chaudhury, S.M., Ghiasi, K., Ranhotra, G.,Seitz, L.M., and Sebti, S. (1980). Nine international breads from sound andhighly-field-sprouted Pacific Northwest soft white wheat. Baker’s Digest 54:22-27.

Foster, N.R., Burchett, L.A., and Paulsen, G.M. (1998). Seed quality of hard redwheat after incipient preharvest sprouting. Journal of Applied Seed Production16: 87-91.

Fretzdorff, B. (1993). Lipolytic enzyme activities in germinating wheat, rye andtriticale. In Walker-Simmons, M.K. and Reid, J.L. (Eds.), Pre-Harvest Sproutingin Cereals 1992 (pp. 270-277). St. Paul, MN: American Association of CerealChemists.

Fretzdorff, B. and Betsche, T. (1998). Is oxalate oxidase indicative of pre-harvestsprouting related deterioration in cereal grains? In Weipert, D. (Ed.), Eighth In-ternational Symposium on Pre-Harvest Sprouting in Cereals (pp. 119-122).Detmold, Germany: Association of Cereal Research.

Gale, M.D. (1989). The genetics of preharvest sprouting in cereals, particularly inwheat. In Derera, N.F (Ed.), Preharvest Field Sprouting in Cereals (pp. 85-110).Boca Raton, FL: CRC Press.

Han, F., Ullrich, S.E., Clancy, J.A., Jitkow, V., Kilian, A., and Romagosa, I. (1996).Verification of barley seed dormancy loci via linked molecular markers. Theo-retical and Applied Genetics 92: 87-91.

Heyne, E.G. (1956). Earl G. Clark, Kansas farmer wheat breeder. Transactions ofthe Kansas Academy of Science 59: 391-404.

Hilhorst, H.W.M. and Toorop, P.E. (1997). Review on dormancy, germinability,and germination in crop and weed seeds. Advances in Agronomy 61: 111-165.

Jensen, S.A. and Heltved, F. (1982). Visualization of enzyme activity in germinat-ing cereal seeds using a lipase sensitive fluorochrome. Carlsberg ResearchCommunications 47: 297-303.

Jones, B.L. and Wrobel, R. (1993). The endoproteinases of germinating barley. InWalker-Simmons, M.K. and Reid, J.L. (Eds.), Pre-Harvest Sprouting in Cereals1992 (pp. 262-269). St. Paul, MN: American Association of Cereal Chemists.

Kato, K., Nakamura, W., Tabiki, T., Mura, H., and Sawada, S. (2001). Detection ofloci controlling seed dormancy on group 4 chromosomes of wheat and compara-tive mapping with rice and barley genomes. Theoretical and Applied Genetics102: 980-985.

Kermode, A.R. (1990). Regulatory mechanisms involved in the transition from seeddevelopment to germination. Critical Reviews in Plant Sciences 9: 155-195.

King, R.W. (1989). Physiology of sprouting resistance. In Derera, N.F (Ed.),Preharvest Field Sprouting in Cereals (pp. 27-60). Boca Raton, FL: CRC Press.

Koeltzow, D.E. and Johnson, A.C. (1993). Comparison of sprout damage analysistechniques. In Walker-Simmons, M.K. and Reid, J.L. (Eds.), Pre-HarvestSprouting in Cereals 1992 (pp. 391-399). St. Paul, MN: American Associationof Cereal Chemists.

Kruger, J.E. (1989). Biochemistry of preharvest sprouting in cereals. In Derera, N.F(Ed.), Preharvest Field Sprouting in Cereals (pp. 61-84). Boca Raton, FL: CRCPress.

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Kruger, J.E. and Hatcher, D.W. (1993). Comparison of newer methods for the deter-mination of -amylase in wheat or wheat flour. In Walker-Simmons, M.K. andReid, J.L. (Eds.), Pre-Harvest Sprouting in Cereals 1992 (pp. 400-408). St. Paul,MN: American Association of Cereal Chemists.

Kruger, J.E., Hatcher, D.W., and Dexter, J.E. (1995). Influence of sprout damage onOriental noodle quality. In Noda, K. and Mares, D.J. (Eds.), Seventh Interna-tional Symposium on Pre-Harvest Sprouting in Cereals 1995 (pp. 9-18). Osaka,Japan: Center for Academic Societies.

Kulp, K., Roewe-Smith, P., and Lorenz, K. (1983). Preharvest sprouting of winterwheat: I. Rheological properties of flours and physicochemical characteristics ofstarches. Cereal Chemistry 60: 355-359

Lin, S.Y., Sasaki, T., and Yano, M. (1998). Mapping quantitative trait loci control-ling seed dormancy and heading date in rice, Oryza sativa L., using backcross in-bred lines. Theoretical and Applied Genetics 96: 997-1003.

Liu, X., Wang, G., Jin, Y., Yang, S., and Li, Y. (1998). Endogenous hormone activ-ity during grain filling of wheat genotypes differing in pre-harvest sprouting. InWeipert, D. (Ed.), Eighth International Symposium on Pre-Harvest Sprouting inCereals 1998 (pp. 99-101). Detmold, Germany: Association of Cereal Research.

Lorenz, K., Roewe-Smith, P., Kulp, K., and Bates, L. (1983). Preharvest sproutingof winter wheat: II. Amino acid composition and functionality of flour and flourfractions. Cereal Chemistry 60: 360-366.

Lorenz, K. and Valvano, R. (1981). Functional characteristics of sprout-damagedsoft white wheat flours. Journal of Food Science 46: 1018-1020.

Lunn, G.D., Major, B.J., Kettlewell, P.S., and Scott, R.K. (2001). Mechanisms lead-ing to excess alpha-amylase activity in wheat (Triticum aestivum, L.) grain in theU.K. Journal of Cereal Science 33: 313-329.

Mares, D.J. (1989). Preharvest sprouting damage and sprouting tolerance: Assaymethods and instrumentation. In Derera, N.F (Ed.), Preharvest Field Sproutingin Cereals (pp. 129-170). Boca Raton, FL: CRC Press.

Mares, D.J. (1998). The seed coat and dormancy in wheat grains. In Weipert, D.(Ed.), Eighth International Symposium on Pre-Harvest Sprouting in Cereals1998 (pp. 77-81). Detmold, Germany: Association of Cereal Research.

Mares, D.J. and Mrva, K. (1993). Late maturity -amylase in wheat. In Walker-Simmons, M.K. and Reid, J.L. (Eds.), Pre-Harvest Sprouting in Cereals 1992(pp. 178-184). St. Paul, MN: American Association of Cereal Chemists.

McCrate, A.J., Nielson, M.T., Paulsen, G.M., and Heyne, E.G., (1981). Preharvestsprouting and -amylase activity in hard red and hard white winter wheatcultivars. Cereal Chemistry 58: 424-428.

McDonald, M.B. (1994). Seed germination and seedling establishment. In Boote,K.J., Bennett, J.M., Sinclair, T.R., and Paulsen, G.M. (Eds.), Physiology and De-termination of Crop Yield (pp. 37-60). Madison, WI: ASA-CSSA-SSSA.

McMaster, G.J., Tomlinson, D., Edwards, R., Ross, A., and Moss, H.J. (1989).Endoprotease activity in rain-damaged Australian wheats. In Ringlund, K.,Mosleth, E., and Mares, D.J. (Eds.), Fifth International Symposium on Pre-harvest Sprouting in Cereals (pp. 65-74). Boulder, CO: Westview Press.

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Miyamoto, T., Tolbert, N.E., and Everson, E.H. (1961). Germination inhibitors re-lated to dormancy in wheat seeds. Plant Physiology 36: 739-746.

Morris, C.F., Moffatt, J.M., Sears, R.G., and Paulsen, G.M. (1989). Seed dormancyand responses of caryopses, embryos, and calli to abscisic acid in wheat. PlantPhysiology 90: 643-647.

Morris, C.F., Mueller, D.D., Faubion, J.M., and Paulsen, G.M. (1988). Identifica-tion of L-tryptophan as an endogenous inhibitor of embryo germination in whitewheat. Plant Physiology 88: 435-440.

Mrva, K. and Mares, D. (1998). Co-ordinated synthesis of high pI alpha-amylaseisozymes in the aleurone of wheat grains. In Weipert, D. (Ed.), Eighth Interna-tional Symposium on Pre-Harvest Sprouting in Cereals 1998 (pp. 131-135).Detmold, Germany: Association of Cereal Research.

Murphy, J.B. and Noland, T.L. (1982). Temperature effects on seed imbibition andleakage mediated by viscosity and membranes. Plant Physiology 69: 428-431.

Muthukrishnan, S., Chaudra, G.R., and Maxwell, E.S. (1983). Hormonal control of-amylase gene expression in barley. Journal of Biological Chemistry 258:

2370-2375.Nagao, S. (1995). Detrimental effect of sprout damage on wheat flour products. In

Noda, K. and Mares, D.J. (Eds.), Seventh International Symposium on Pre-Har-vest Sprouting in Cereals (p. 3-8). Osaka, Japan: Center for Academic Societies.

Nielsen, M.T., McCrate, A.J., Heyne, E.G., and Paulsen, G.M. (1984). Effect ofweather variables during maturation on preharvest sprouting of hard white win-ter wheat. Crop Science 24: 779-782.

Paterson, A.H. and Sorrells, M.E. (1990). Inheritance of grain dormancy in white-kerneled wheat. Crop Science 30: 25-30.

Paulsen, G.M. (1985). Technology for improvement and production of wheat inChina. Journal of Agronomic Education 14: 63-68.

Peiper, H.J. (1998). Enzyme potential of cereals suitable for the production of etha-nol and glucose syrup. In Weipert, D. (Ed.), Eighth International Symposium onPre-Harvest Sprouting in Cereals 1998 (pp. 56-66). Detmold, Germany: Associ-ation of Cereal Research.

Shakeywich, C.F. (1973). Proposed method of measuring swelling pressure ofseeds prior to germination. Journal of Experimental Botany 24: 1056-1061.

Smith, J.D. and Fong, F. (1993). Classification and characterization of the vivipa-rous mutants of maize (Zea mays L.). In Walker-Simmons, M.K. and Reid, J.L.(Eds.), Pre-Harvest Sprouting in Cereals 1992 (pp. 295-302). St. Paul, MN:American Association of Cereal Chemists.

Stahl, M. and Steiner, A. (1998). Viability loss of sprouted wheat seeds during stor-age. In Weipert, D. (Ed.), Eighth International Symposium on Pre-HarvestSprouting in Cereals 1998 (pp. 123-130). Detmold, Germany: Association ofCereal Research.

Upadhyay, M. and Paulsen, G.M. (1988). Heritabilities and genetic variation forpreharvest sprouting in progenies of Clark’s Cream white winter wheat. Euphy-tica 38: 93-100.

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Vertucci, C.W. and Leopold, A.C. (1986). Physiological activities associated withhydration levels in seeds. In Leopold, A.C. (Ed.), Membranes, Metabolism, andDry Organisms (pp. 35-49). Ithaca, NY: Comstock Publishing Association.

Walker-Simmons, M.K. (1987). ABA levels and sensitivity in developing wheatembryos of sprouting resistant and susceptible cultivars. Plant Physiology 84:61-66.

Walker-Simmons, M.K., Aurelio-Cadenas, A., Verhey, S.D., Zhang, P., Holappa,L.D., and David Ho, T.-H. (1998). Involvement of PKABA/protein kinase inmolecular control of -amylase and protease gene expression in barley aleuronecells. In Weipert, D. (Ed.), Eighth International Symposium on Pre-HarvestSprouting in Cereals 1998 (pp. 203-217). Detmold, Germany: Association ofCereal Research.

Wellington, P.S. (1956). Studies on the germination of cereals: 2. Factors determin-ing the germination behaviour of wheat grains during maturation. Annals of Bot-any 20: 481-486.

Wu, J. and Carver, B.F. (1999). Sprout damage and preharvest sprout resistance inhard white winter wheat. Crop Science 39: 441-447.

Yamaguchi, J., Toyofuku, K., Morita, A., Ikeda, A., Matsukura, C., and Perata, P.(1998). Sugar repression of alpha-amylase genes in rice embryos. In Weipert, D.(Ed.), Eighth International Symposium on Pre-Harvest Sprouting in Cereals1998 (pp. 136-145). Detmold, Germany: Association of Cereal Research.

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Chapter 7

The Exit from Dormancy and the Induction of GerminationThe Exit from Dormancyand the Induction of Germination:

Physiological and Molecular Aspects

Rodolfo A. SánchezR. Alejandra Mella

INTRODUCTION

Timing and location of germination are crucial for the chances of successof the newly produced plant, and, accordingly, the temporal and spatial pat-terns of germination of the seeds of many species are finely tuned to the en-vironmental scenario. Dormancy plays a central role in the adjustment ofthe behavior of seed populations to the restrictions and opportunities of agiven environment (Chapter 8).

Dormancy is a physiological condition that prevents germination in anotherwise favorable set of external conditions (for a more detailed discus-sion of the definition of dormancy see Chapter 8). At first sight it may seemparadoxical that a sophisticated mechanism evolved in seeds to block theironly function. However, it takes only a very brief inspection of the conse-quences of the lack of dormancy to appraise its importance. Were it not fordormancy, the seeds of many species would germinate when still attachedto the parent plant, not surviving to be established in the soil, or the seed-lings of an annual summer weed would be produced at the end of fall and sobe condemned to die because of the cold of winter. The varied aspects of therelationships between dormancy and population dynamics of many specieshave been treated in excellent books such as those by Bewley and Black(1994) and Baskin and Baskin (1998).

The level of dormancy varies with time, provoking changes in the sensi-tivity of germination to various environmental factors (Chapter 8). At cer-tain times dormancy is low enough and allows that a certain environmentalfactor (i.e., light, temperature, nitrates, or combinations of them) can termi-nate dormancy and induce germination. For seeds in soil, these factors rep-

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resent important signals carrying essential information, cueing germinationto the proper environmental situation.

Once the proper signal is perceived, a series of processes are set in motionthat finally result in reactivation of embryo growth and radicle protrusionthrough the covering structures. Expansion of the embryonic axis requireschanges in the embryo as well as in the surrounding tissues. In this chapterwe will consider the physiological and molecular aspects of these pro-cesses. A significant proportion of the physiological research has been car-ried out with seeds of lettuce (Bewley and Halmer, 1980/1981; Borthwickand Robbins, 1928; Carpita, Ross, and Nabors, 1979; Carpita et al., 1979;Psaras and Georghiu, 1983; Psaras, Georghiu, and Mitrakos, 1981) andsome Solanaceae species (tomato, pepper, Datura) (Dahal, Nevins, andBradford, 1996; de Miguel et al., 2000; de Miguel and Sánchez, 1992;Groot and Karssen, 1987, 1992; Groot et al., 1988; Mella, Maldonado, andSánchez, 1995; Ni and Bradford, 1993; Nonogaki and Morohashi, 1996,1999; Nonogaki, Nomaguchi, and Morohashi, 1995, 1998; Sánchez and deMiguel, 1985, 1997; Sánchez et al., 1990; Watkins and Cantliffe, 1983;Watkins et al., 1985), although most of our knowledge of the molecular andgenetic aspects of germination has been derived mainly from work withseeds of Arabidopsis, tomato, and tobacco (Bradford et al., 2000; Grappinet al., 2000; Karssen and Lacka, 1986; Koornneef and van der Veen, 1980).Several of these aspects have been covered by excellent reviews (Bewley,1997; Hilhorst, 1995; Koornneef, Bentsink, and Hilhorst, 2002; Peng andHarberd, 2002). Since in almost all of these species germination is con-trolled by light, this chapter will focus in the light control of germination inspecies with coat-imposed dormancy. In seeds with this type of dormancy,germination depends on the balance between the expansive capacity of theembryo and the restrictions imposed by the surrounding tissues. Light per-ception by the photoreceptors may affect processes in components of thebalance, initiating or blocking germination. In the following sections wewill describe, after a brief description of the known photoreceptors, thechanges in the embryo and the surrounding tissues (mainly the endosperm)related to germination.

THE EFFECTS OF LIGHT PHOTORECEPTORS

Light can promote or inhibit germination depending on its spectral com-position and irradiance, the physiological status of the seeds, and the condi-tions of the other environmental factors, particularly temperature and waterpotential (Bewley and Black, 1994; Casal and Sánchez, 1998).

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Promotion of germination by light has so far been found to be mediatedonly by the phytochromes. The loss of dormancy associated with certain sit-uations found in the soil, or some incubation conditions in controlled envi-ronments, cause some seeds to display an extreme sensitivity to light (Casaland Sánchez, 1998). In those cases, millisecond exposures to sunlight aresufficient to induce germination; this is known as a very low fluence re-sponse (VLFR), which in Arabidopsis is mediated by phytochrome A(Botto et al., 1996; Shinomura et al., 1996). This response is saturated withvery low levels of Pfr form of phytochrome (often less than 1 percent of to-tal phytochrome [Pt] in the Pfr form) and does not display the classical redlight (R)–far-red (FR) light reversibility (Casal, Sánchez, and Botto, 1998;Mandoli and Briggs, 1981). This mode of action of the phytochromes al-lows the detection of the brief exposure to light the seeds experience duringsoil disturbances such as those occurring during agricultural tillage opera-tions (Scopel, Ballaré, and Sánchez, 1991).

In other physiological conditions, termination of dormancy requireslight establishing Pfr/Pt > 0.05, and the photocontrol of germination dis-plays the classical R-FR reversibility (Borthwick et al., 1952). This is calledthe low fluence response (LFR). When influenced by this mode of action,germination depends on the R:FR ratio of the light reaching the seeds,which is a good signal of the density of the canopy covering the soil(Insausti, Soriano, and Sánchez, 1995; Vazquez-Yañez et al., 1996; Vázquez-Yañez and Smith, 1982). The photoreceptors of this mode of action identi-fied in Arabidopsis seeds are phytochromes B and E (Hennig et al., 2001).Almost all the research so far published on the physiological and molecularprocesses involved in the promotion of germination by light has been donewith seeds displaying the LFR.

Inhibition of germination can be produced by light in the FR or blue (B)spectral regions (Bewley and Black, 1978). With few exceptions, inhibitionby FR requires prolonged exposures to continuous light ( max 710 to 720nm) or very frequent pulses and is irradiance dependent (Hartmann, 1966;Mancinelli, 1980). This effect is mediated by the high irradiance response(HIR) mode of action of phytochrome. In tomato seeds it has been shownthat phyA is the photoreceptor (Shichijo et al., 2001). The HIR can both in-hibit germination of dark-germinating seeds and antagonize the promotionof germination initiated by an LFR or a VLFR (Burgin et al., 2002; deMiguel et al., 1999). A continuous FR treatment can block germinationeven if given many hours after the R pulse starting the LFR (even very closeto the time of radicle emergence). A subsequent pulse of R can relieve theinhibition imposed by the continuous FR treatment; therefore, the possibil-ity of the antagonism LFR-HIR is there for much of the duration of the ger-mination process (de Miguel et al., 2000). Blue light can also inhibit germi-

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nation of many species, and R antagonizes its effect (Gwynn and Schiebe,1972; Malacoste et al., 1972). The photoreceptor for the blue light action inseeds has not been identified so far.

It is clear that the control of germination is influenced by different photo-receptors interacting in a variety of ways. We are only beginning to under-stand the complex set of coordinated physiological and biochemical eventsinvolved in the photocontrol of germination and their regulation by crosstalk between transduction chains initiated by endogenous and environmen-tal signals.

EMBRYO GROWTH POTENTIAL

The promotion of germination is commonly associated with an increasein embryo growth potential. Although in seeds with coat-imposed dor-mancy the isolated embryos can grow without any particular stimulus, it hasbeen observed that the embryos from seeds that are less dormant or havebeen exposed to a promotive treatment (i.e., light) have a greater growthrate than those from nonstimulated seeds (Figure 7.1a and b). The differ-ence in growth potential is more evident when the incubation medium con-tains a factor opposing embryo expansion, such as an osmoticum. Growthof embryos isolated from lettuce (Carpita, Ross, and Nabors, 1979; Carpitaet al., 1979) and Datura ferox seeds (de Miguel and Sánchez, 1992) is pro-moted by an R pulse, and the promotion by R is reversed if immediately fol-lowed by an FR pulse, displaying a typical LFR (Figure 7.2). Abscisic acid(ABA) antagonizes the LFR promotion of growth potential, whereas exoge-nous gibberellins (GAs) enhance growth potential in dark-incubated seeds(Karssen and Lacka, 1986). On the other hand, lowering the external waterpotential interacts with phytochrome in a complex fashion; small reduc-tions in water potential enhance the phytochrome promotion of embryogrowth potential, whereas large reductions block the LFR (de Miguel andSánchez, 1992).

Although, as discussed earlier, an increase in the embryo growth poten-tial accompanies the stimulus of germination, and the available evidencesupports the contention that it may be necessary for radicle protrusion, thephysiological and molecular bases of the enhancement in the embryo’s ex-pansion capacity are poorly understood. In the pioneering work of Carpitaand colleagues (1979) with lettuce seeds it was shown that phytochrome-mediated K+ transport could led to a decrease in the osmotic potential suffi-cient to explain a good part of the growth response. However, as the authorspointed out, it was likely that a change in the cell wall extensibility mightalso be involved. That modification of wall extensibility plays a predomi-

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nant role in ABA inhibition of embryo growth and germination is supportedby studies with Brassica napus seeds (Schopfer and Plachy, 1985). The pos-sibility that wall extensibility changes may participate in the expansion ca-pacity of the embryo is also suggested by changes in expression of expansingenes. The temporal and spatial pattern of expression of two expansin genesin tomato seeds, LeEXP8 and LeEXP10, are consistent with a role forexpansins, and GA promotes the transcription of both genes in gibberellin-deficient gib1 seeds (Chen, Dahal, and Bradford, 2001). On the other hand,low water potential blocks expression of LeEXP8, which is also inhibitedby ABA (Chen, Dahal, and Bradford, 2001). Expansin transcript levels arealso increased by R in the embryos of D. ferox seeds, an FR-reversible effectthat is in good agreement with the photocontrol of germination (Mella et al.,2004). Although these results do not establish a direct causal connection be-

FIGURE 7.1 The effect of ABA on embryo length frequency distribution. Seedswere incubated in water or 100 µM ABA for 46 h after R. At the end of the incuba-tion the seeds were detipped, irradiated with FR, and transferred to water. Afterfurther 24h incubation at 25°C in the dark, embryo length was measured.Theseed pool in (a) has a higher degree of dormancy (12 months of dry storage)than seeds in (b) ( 24 months in dry storage). Data from ABA-treated seeds in (b)could not be fitted to a normal distribution.Assuming two subpopulations with dif-ferent sensitivity to ABA allowed a good fit. (Source: de Miguel, L., Burgin, J.,Casal, J. J., and Sánchez, R. A., unpublished results.)

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tween expansin gene expression and embryo growth potential, they cer-tainly support an intervention of expansins in this process.

Taken together, the results obtained with seeds of different species sug-gest that the increase in embryo growth potential associated with germina-tion may include a decrease in osmotic potential, consequently a rise inturgor pressure, and an increase in cell wall extensibility, probably with theparticipation of expansins.

ENDOSPERM WEAKENING

In seeds whose embryos are completely surrounded by a rigid endo-sperm, radicle emergence requires a significant reduction in the physical re-striction that the endosperm opposes to embryo expansion. In particular, themicropylar endosperm (named the endosperm cap), which is the region di-rectly opposed to the embryo radicle, must be weakened. Endosperm capweakening has been shown to precede radicle protrusion (Groot and Kars-sen, 1987; Sánchez et al., 1990; Watkins and Cantliffe, 1983) (Figure 7.3)and is accompanied by extensive structural changes. In addition to changesin the cell walls, profound alterations are produced in other parts of thecells, protein and lipid reserves are degraded, and extensive vacuolationtakes place; the structure changes from that typical of reserve cells to meta-bolically active ones (Figures 7.4 and 7.5) (Mella, Maldonado, and Sánchez,

– – – –

FIGURE 7.2. Growth in osmotica of radicles from R- and FR-treated lettuceembryos. ‘Grand Rapids’ lettuce seeds were exposed to red (closed squares) orfar-red (open squares) light. The embryos were removed and placed in solutionsof polyethylene glycol of different osmotic strengths and the growth rate over 24h was determined. (Source: Adapted from Carpita et al., 1979.)

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1995; Psaras, Georghiu, and Mitrakos, 1981; Sánchez et al., 1990). Thispreradicle protrusion syndrome is restricted to the micropylar region; therest of the endosperm (frequently called the lateral endosperm) remains un-changed.

In relation to weakening, the attention has been focused on changes inthe cell walls. The reduction in the mechanical resistance of the endospermcap has been associated with the hydrolysis of cell wall polysaccharides,primarily of the main component: a mannose polymer, probably a -(1,4)-mannan (Table 7.1) (Sánchez et al., 1990). A large increase in the activity ofmannan-degrading enzymes precedes radicle protrusion when germinationis promoted by light, through a LFR of the phytochromes, or GA in tomato(Groot and Karssen 1987; Nonogaki and Morohashi, 1996) and D. ferox(de Miguel and Sánchez 1992; Sánchez et al., 1990) seeds. In tomato seeds,a endo- -mannanase gene (LeMAN2) is exclusively expressed in the endo-sperm cap prior to radicle emergence (Nonogaki, Gee, and Bradford, 2000).Consistently, red light strongly increases, in an FR-reversible fashion, thetranscript level of DfMAN1 (which shows high homology with LeMAN2) inD. ferox seeds only in the endosperm cap (Burgin et al., 2000). Although di-rect evidence is not yet available, the information from tomato and Daturastrongly supports the possibility that cell wall mannan degradation is part ofthe mechanism of endosperm cap softening (Figure 7.6). This does not im-ply that mannan degradation or high mannanase activity is sufficient for en-dosperm softening or, even less, for germination. As described in the previ-ous section, inhibiting embryo expansion may by itself block germinationeven in cases when the surrounding tissues offer little or no resistance toembryo growth (Schopfer and Plachy, 1993). Endosperm weakening mayalso involve changes in cell wall components other than mannose polymers.Several hydrolases (xyloglucan endotransglycosylase, arabinosidase, etc.)

FIGURE 7.3. Photographs of the external aspect of the micropylar region ofdecoated Datura ferox L. seeds. (A) 46 h after a noninductive far-red pulse; (B)46 h after an inductive R pulse (red light) showing signs of weakening; (C) germi-nated seed, with the protruded radicle and broken micropylar endosperm.

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have been found to be expressed during tomato germination and could con-tribute to cell separation (Bradford et al., 2000). In tobacco seeds, a goodcorrelation has been found between the increase in the activity of a class I

-1,3-glucanase and the promotion of germination. The gene coding forthat glucanase is expressed specifically in the endosperm cap, and its over-expression alleviates the inhibitory action of ABA on tobacco seeds(Leubner-Metzger, Fründt, and Meins, 1996; Leubner-Metzger et al., 1995).However, it has been shown in tomato seeds that endosperm cap weakeningbegins before the expression of the -1,3-glucanase gene can be detectedand although ABA effectively inhibited -1,3-glucanase gene expression, itdid not affect endosperm softening (Wu et al., 2001). In the same work no

FIGURE 7.4. Scanning electron micrographs of median sections of micropylarand adjacent bulk endosperm of Datura seeds glutaraldehyde-osmium fixed andcritical point dried. (A, C) FR-irradiated seeds; (B, D) FR+R-irradiated seeds;sampling at 38 hours after irradiation. Micrographs C and D are enlargementscorresponding to the encircled areas of A and B. (Source: After Sánchez et al.,1990. Reprinted with permission of National Research Council of Canada.)

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evidence was found of a substrate for the enzyme in endosperm cap cellwalls; therefore, the involvement of the -1,3-glucanase in the weakeningprocess in tomato seeds was not supported and other functions for this en-zyme were considered more likely. In addition, it has been suggested thatexpansins could play a role in endosperm weakening either by facilitatingthe access of hydrolases to their substrates or by loosening hemicellulosicbonds (Bradford et al., 2000; Chen, Dahal, and Bradford, 2001). This prop-osition is supported by the results of Chen, Dahal, and Bradford (2001)showing that a specific expansin gene, LeEXP4, is expressed exclusivelyin the tomato endosperm cap prior to radicle emergence and is up-regulatedby GA.

Low water potential prevents endosperm weakening (Chen and Brad-ford, 2000; de Miguel and Sánchez, 1992; Sanchez et al., 2002). In D. feroxseeds it has been shown that germination is inhibited by water potentialsthat do not reduce phytochrome promotion of embryo growth potential butprevent endosperm weakening (de Miguel and Sánchez, 1992). Therefore,

FIGURE 7.5. Median and longitudinal sections of the micropylar portions ofDatura ferox L. seeds, 48 h after R light (A, B ) or FR light (C, D) treatment. (A, C)low magnification view—the arrows in (A) indicate the zone with extensive deg-radation of protein bodies; (B) an enlargement of a portion between the arrows—note the vacuolation of protein bodies; (D) view of an area with similar localiza-tion of (B) but in a FR-treated seed—note the abundance of storage material andthe absence of vacuoles. (Source: After Mella, Maldonado, and Sánchez, 1995,© American Society of Plant Biologists. Reprinted with permission.)

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TABLE 7.1. Sugar composition of cell wall polysaccharides in the micropylar por-tion of the endosperm of Datura ferox L. seeds induced to germinate andnoninduced controls

R FR

(mg/micropylar portion)Rha 0.5 0.35Rib 0.025 0.04Ara 4.4 3.2Xyl 0.6 0.6Man 11.2 3.3Gal 1.7 1.1Glc 0.9 0.65Uronic acid 2.3 1.75Cell 4.2 3.3

Source: Adapted from Sánchez et al., 1990, © American Society of Plant Biolo-gists. Reprinted with permission. Note: Sampling was made 38 h after irradia-tion. The data are averages of two determinations.

Imbibition time (h)

Pun

ctur

efo

rce

(N)

12 24 36 48 600.4

0.6

0.8

1.0

1.2

FIGURE 7.6. Puncture force analysis of wild-type (‘Money Maker’) seeds inwater (closed circles) and gib-1 mutant seeds in water (open squares) and 100 mMGA (closed squares).Error bars indicate standard errors (n = 24) when larger thanthe symbols. (Source: Chen and Bradford, 2000, © American Society of PlantBiologists. Reprinted with permission.)

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low water potential, at least at certain values, can prevent the induction ofgermination mainly through its effect on endosperm softening. The mecha-nisms involved, however, are not clear yet. A relationship does exist be-tween low water potential effects on the phytochrome-induced reduction inmannose content of the cell walls and endosperm cap weakening in D. feroxseeds (Sánchez et al., 2002); in addition, the inclusion of an osmoticum inthe incubation medium reduced the release of mannose by tomato endo-sperm caps (Dahal, Nevins, and Bradford, 1997). These results suggest thatinterference with mannan degradation could be one of the ways throughwhich water availability influences endosperm resistance to embryo pene-tration. On the other hand, low water potential does not prevent the increasein mannanase activity in phytochrome-promoted D. ferox (Sanchez et al.,2002) or in tomato seeds (although see Toorop, van Aelst, and Hilhorst,1998). In D. ferox low water potential inhibits the increase in mannosidaseassociated with germination (and this could indirectly hamper mannan deg-radation), whereas in tomato it has been found that LeEXP4 expression isreduced proportionally to the inhibition of endosperm cap weakening(Chen and Bradford, 2000). If LeXP4 facilitates the access of the hydrolasesto their substrates, inhibiting its production may be an obstacle to weaken-ing, even if the activity of some of the hydrolases (i.e., mannanase) remainshigh. It seems likely, then, that a restriction in water supply may interferewith endosperm weakening, down-regulating the genes encoding some ofthe proteins contributing to the cell wall changes, but not necessarily affect-ing all of them.

Endosperm cap weakening is also blocked when the promotion of germi-nation by an LFR of the phytochromes is antagonized by exposure to con-tinuous FR through an HIR (de Miguel et al., 2000; Mella et al., 2002).Since the HIR does not affect embryo growth potential, it should influencegermination interfering with endosperm cap softening. The continuous FRtreatment can block germination of a part of the seed population even if it isapplied when endosperm weakening has already advanced perceptibly. In-terestingly, in a similar part of the population it provokes a sharp reductionin mannanase activity (Figure 7.7) (down to the values typical ofnoninduced seeds) and the interruption of the weakening process. The HIRcan disengage the weakening process even at relatively advanced stage, but,apparently, the antagonism does not involve every process promoted by theLFR, and even some of the ones affected may be influenced in differentways by each of the phytochromes’ modes of action. While the LFR pro-motes mannanase activity and increases the transcripts level of DfMAN1and DfEXP2 in D. ferox seeds, the HIR inhibits mannanase activity but doesnot modify the transcript levels of neither of them (Burgin et al., 2000;Mella et al., 2004).

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–24 h 0 h 45 h

51 h

50

0.1 1

50

FRp

FRcR

0 h 45 h

51 h

50 50

0.1

50 50

Mannanase

Frac

tion

ofth

epo

pula

tion

activity

0 h 45 h

51 h

0.

0

0 h 45 h

51 h

0.

0

0 h 45 h

51 h

0 %

70 %

100 %

40 %

Preincubation

FR

FRp

FRc

R

0 h 45 h

51 h

Germination count 72 h

..

100 100

10 100

1000

10000

100 100

10.1

10 001 0100

01000FR

FIGURE 7.7. Endo- -mannanase activity measured seed by seed in the popula-tion of Datura ferox induced by LFR or by LFR-HIR. R light promotes germinationand the activity of endo- -mannanase. In the noninduced seeds treated with FRimmediately after R (FR) the values are substantially lower than in those seedsinduced by R (R). The activity was evaluated 45 h after light treatments in dark-ness (20 to 30°C) (FRp). When an FR pulse is given 45 h after R (FR pulse 3min·h–1, 300 mmol·m–2·s–1), and the activity is measured 6 h later, mannanaseactivity decreases in part of the population. The endo- -mannanse activity wasevaluated 51 h after initial light treatment (FRc). If instead of a pulse, FR is givencontinuously during 6 h (15 mmo·l–2·s–1), mannanase activity decreased in alarger fraction of seeds. The endo- -mannanase activity was evaluated 51 hafter initial light treatment. (Source: Modified from Mella et al., 2002).

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Whether the inhibition of germination by ABA includes an effect on en-dosperm softening is not completely clear. Although some experimentsshow that part of endosperm cap weakening is blocked by ABA in tomatoseeds (Toorop, van Aelst, and Hilhorst, 2000), in other studies, with thesame genotype and similar methods, no effect of ABA was found (Chen andBradford, 2000). So far none of the physiological steps that are thought tobe related to endosperm softening, such as mannanase and LeEXP4, are af-fected by exogenous applications of ABA; if there is an ABA-sensitivephase in the weakening process it depends on a process that has so fareluded us (Chen and Bradford, 2000; Nonogaki, Gee, and Bradford, 2000).

Although we do not yet have definite knowledge on the process of endo-sperm softening, it is apparent that it may depend on a variety of mecha-nisms and not every regulatory factor affects all of them.

Other tissues that restrict the expansion of the embryos are the testa, asshown in Arabidopsis (Debeaujon and Koornneef, 2000), and the perispermof muskmelon (Welbaum et al., 1995).

TERMINATION OF DORMANCY: ITS RELATIONSHIPWITH THE SYNTHESIS AND SIGNALING

OF GIBBERELLINS AND ABA

The paramount importance of GA for germination has been recognizedfor a long time (Bewley and Black, 1994) and is most clearly demonstratedin studies with mutants of Arabidopsis and tomato that are severely GA de-ficient (Groot and Karssen, 1987; Koornneef and van der Veen, 1980). Theinduction of germination by light requires GA synthesis (Derkx and Kars-sen, 1993; Yang et al., 1995), and in lettuce seeds an R pulse causes a signif-icant increase in GA content; the effect of R is FR-reversible as in the classi-cal LFR (Toyomasu et al., 1993). In addition to increasing the GA content,the reversible R-FR response enhances the sensitivity to GA in seeds (Yanget al., 1995). In lettuce and Arabidopsis, R promotes the expression of genesencoding GA 3- -hydroxylase, a key enzyme in the active GA biosyntheticpathway (Toyomasu et al., 1998; Yamaguchi et al., 1998). In Arabidopsis,two GA 3- -hydroxylase genes, GA4 and GA4H, are under the control ofthe phytochromes. The Pfr of phyB promotes GA4H, whereas GA4 is con-trolled by some other stable phytochrome (Yamaguchi et al., 1998). Whetherpromotion of germination by the VLFR, which in Arabidopsis is mediatedby phyA, is also related to the up-regulation of these genes is still unknown.

Although the increase in GA levels affects processes in both the embryoand the endosperm, the available evidence suggests that the synthesis of GAtakes place only in the embryo. Light promotes changes only when the em-

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bryo is in contact with the endosperm cap (Sánchez and de Miguel, 1997),and the presence of the embryo can be replaced by supplying the endospermcaps with exogenous GA (Groot et al., 1988; Sánchez and de Miguel,1997). Moreover, the transcripts of DfHydrox, a GA 3- -hydroxylase gene,are only found in the embryo and in significantly higher amounts after an Rpulse than when R is immediately followed by FR (Burgin et al., 2000). TheGA synthesized in the embryo would migrate to the endosperm cap where itinduces weakening. In the micropylar endosperm of tomato, GA promotesthe expression of a number of cell wall hydrolases and related proteins:endo- -mannanase, cellulase, arabinosidase, xyloglucan endotransglyco-sylase, expansin, etc. (Bradford et al., 2000). Because the path of GA fromthe embryo to the micropylar endosperm includes an apoplastic segment, itseems likely that the GA may reach other endosperm cells in addition tothose in the micropylar region. However, only the cells of the endospermcap respond to GA (before radicle protrusion), suggesting a greater sensi-tivity of these cells to GA.

Recent work has shown the participation in the control of germination oftwo GA-response regulators: RGL1 and RGL2 (Peng and Harberd, 2002).Both are repressors of GA responses; loss-of-function mutations in RGL2completely restored germination to ga1-3, eliminating the requirement forexogenous GA (Lee et al., 2002). In wt Arabidopsis, RGL2 transcript levelsshow a transitory increase after the initiation of imbibition followed by adecrease with the advance of germination; interestingly, in the GA-defi-cient ga1-3 the RGL2 transcripts remained at high levels throughout the in-cubation period unless exogenous GA was supplied (Lee et al., 2002). It hasalso been shown that SPY (O-GlcNAc transferase) influences seed germi-nation. In Arabidopsis, SPY alleles confer resistance of germination to theGA-synthesis inhibitor paclobutrazol and restore the germination capacityto ga1-2 (Jacobsen and Olszewski, 1993). On the basis of the available evi-dence, Peng and Harberd (2002) have proposed the following scheme. Indormant seeds upon imbibition proteins are expressed that repress germina-tion (Peng and Harberd, 2002). In the proper physiological scenario phyto-chrome activation by light induces GA synthesis. The increased GA leveldown-regulates the expression of repressors such as RGL2 and SPY thatmight increase the germination potential of the embryo. At the same timethe increase in GA reaches the endosperm cap where signaling factors(GCR1, SLY, and CTS are candidates) would induce the expression of theproteins (e.g., mannanase, expansins, XET, etc.) related to weakening. Al-though testing of several of these propositions is still pending, it is consis-tent with most of the information at hand and is useful to guide the design offuture experiments.

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ABA has an essential role in establishing dormancy during seed develop-ment (Hilhorst and Karssen, 1992; Hilhorst, 1995), which influences the re-sponses of mature seeds to various environmental factors. In addition, syn-thesis of ABA during imbibition has been shown to be important forgermination. ABA levels increase upon imbibition different in dormant andnondormant seeds (Grappin et al., 2000; Le Page-Degivry and Garello,1992), and inhibitors of ABA biosynthesis promote germination (Grappinet al., 2000). In the same line are observations of a decline in ABA contentafter a R pulse promoting germination (Tillberg and Björkman, 1993) andthe prevention of the decline of the ABA content normally occurring in ger-minating lettuce seeds by high-temperature treatments inhibitory of germi-nation (Yoshioka, Endo, and Satoh, 1998). Mutations in several geneschange the sensitivity of germination to ABA. The abi and era mutants havereduced and enhanced responses respectively (Koornneef, Bentsink, andHilhorst, 2002), the ethylene insensitive 2 (ein2) and ethylene response (etr)mutants of Arabidopsis are hypersensitive, while the ctr genes have lesssensitivity to ABA. Moreover, ABA and ethylene signaling interact withsugar signaling (Finkelstein, Gampala, and Rock, 2002) and mutants defi-cient (det2-1) or insensitive (bri1-1) to brassinosteroids are also more sensi-tive to ABA, suggesting that the BR signal participates in the control of ger-mination opposing ABA inhibition (Steber and McCourt, 2001). Althoughthe description of the components of the web of signaling networks modu-lated by different regulators is incomplete, it shows the variety and com-plexity of the system that permits the integration of environmental and in-ternal signals which modulate germination.

CONCLUDING REMARKS

Germination is a crucial process for the adjustment of many plant popu-lations to their environment. Taking into account the variety of environmen-tal scenarios and plant genotypes, it is not surprising that the diversity ofexternal factors and their combinations can control seed responses. Germi-nation itself is a complex process involving a number of finely coordinatedchanges in the embryo and the surrounding tissues with the participation ofa large number of genes. Congruent regulation of the several molecular,biochemical, and physiological events leading to germination requirescross talk between endogenous and environmental signals. The elements ofthis dialogue (several phytochromes, GA, ABA, ethylene, BR, sugars, etc.)are part of an intricate network with versatile switches and multiple path-ways. In this context it is to be expected that a particular factor may not al-

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ways have the same relevance and that correlations could vary according tothe physiological and environmental scenario.

In the interplay between the embryo and the enveloping tissues, the roleof the embryo seems to be central. It is in the embryos where the light sig-nals are perceived (and the same is probably the case with other environ-mental signals) and there where GA and likely other regulators are pro-duced that profoundly influence the activity in the surrounding tissues.Particularly when the endosperm is the main barrier to embryo growth, theGA synthesized in the embryo provokes changes in the expression of genescoding for cell wall hydrolases and other proteins involved in weakening.When the testa is limiting embryo expansion, as in Arabidopsis, it is alsothought that testa weakening may depend on GA action (Debeaujon andKoornneef, 2000). Figure 7.8 shows a tentative scheme in which data fromseveral species (we do not have the whole picture in just one species) andsome components of the processes with different degrees of experimentalsupport are included depicting some of the connections between the em-bryo and the endosperm and the points of action of some internal and exter-nal factors. The data available seem to indicate that not all of these factorsinfluence the same processes. No master switch appears to be in control ofthe whole system. Once germination is promoted (e.g., by light) a numberof activities are set in motion in the embryo and the endosperm. The actionof some of the factors which can block that response may affect only part ofthem. For instance, ABA can sharply decrease embryo growth potentialwithout affecting most of the endosperm softening; in contrast, the HIR ofthe phytochromes interferes with endosperm softening but does not seem toaffect embryo growth potential. However, we have still a long way to go be-fore being able to put the central pieces of the puzzle in their proper places.It would be helpful to have integrated information about more than one sys-tem. This is currently limited by the problem that knowledge on certainparts of the system has advanced more in some species than in others and, infact, all the processes and their interactions may not be identical in the dif-ferent species from which most of the information has been gathered so far.

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Y a

MICROPYLAR

ENDOSPERM

WEAKENING

EMBRYO

GROWTH

POTENTIAL

GERMINATION

Light (LFR)

GA 3 b hydroxylase Gene

GA

GA signaling

(RGL-1 and 2 ?)

Phy signaling net

Expansins

GA

Expansins

XET

Mannanase

etc

?

GA signaling

(CTS -SPY?)

(SLY, RGA ?)

?

ABA

ABA signaling

ABA

Light (HIR)

ABA signaling

?

Pr Pfr

�a

MICROPYLAR

ENDOSPERM

WEAKENING

EMBRYO

GROWTH

POTENTIAL

GERMINATION

Light (LFR)

GA 3- -hydroxylase Gene�

GA

GA signaling

(RGL-1 and 2 ?)

Phy signaling net

Expansins

GA

Expansins

XET

Mannanase

etc.

?

GA signaling

(CTS -SPY?)

(SLY, RGA ?)

?

ABA

ABA signaling

ABA

Light (HIR)

ABA signaling

?

Pr PfrPr Pfr

FIGURE 7.8. Diagramatic representation of part of the molecular signaling andcomponents acting on the termination of coat-imposed dormancy and the induc-tion of germination. Broken lines and question marks represent probable butunconfirmed interactions.

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REFERENCES

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Bewley, J.D. (1997). Breaking down the walls—A role for endo mannanase in re-lease from seed dormancy? Trends in Plant Science 2: 464-469.

Bewley, J.D. and Black, M. (1978). The Physiology and Biochemistry of Seeds inRelation to Germination, Volume 1. New York: Springer-Verlag.

Bewley, J.D. and Black, M. (1994). Seeds: Physiology of Development and Germi-nation, Second edition. New York: Plenum Press.

Bewley, J.D. and Halmer, P. (1980/1981). Embryo-endosperm interactions in thehydrolysis of lettuce seeds reserves. Israel Journal of Botany 29: 118-132.

Borthwick, H.A., Hendricks, S.B., Parker, M.W., Toole, E.H., and Toole, V.K.(1952). A reversible photoreaction controlling seed germination. Proceedings ofthe National Academy of Sciences, USA 38: 662-666.

Borthwick, H.A. and Robbins, W.W. (1928). Lettuce seed and its germination.Hilgardia 3: 275-305.

Botto, J.F., Sánchez, R.A., Whitelam, G.C., and Casal, J.J. (1996). Phytochrome Amediates the promotion of seed germination by very low fluences of light andcanopy shade light in Arabidopsis. Plant Physiology 110: 439-444.

Bradford, K.J., Chen, F., Cooley, M.B., Dahal, P., Downie, B, Fukunaga, K.K., Gee,O.H., Gurusinghe, S., Mella, R.A., Nonogaki, H., et al. (2000). Gene expressionprior to radicle emergence in imbibed tomato seed. In M. Black, K.J. Bradford,and J. Vazquez-Ramos (Eds.), Seed Biology: Advances and Applications (pp. 231-251). New York: CABI Publishing.

Burgin, M., Arana, V., de Miguel, L., Staneloni, R., and Sanchez, R. (2002). Re-sponses of Datura ferox seeds to far red light pulse are inhibited by continuousfar red. VII International Workshop on Seed Biology (p. 113), Salamanca, Spain,May 12-16.

Burgin, M.J.P.F.L., Mella, A., Staneloni, R., and Sánchez, R.A. (2000). The tran-scription of endo- -mannanase and GA 3 -hydroxylase genes of Datura feroxseeds is regulated by phytochrome. Report No. 146. Presented at Plant Biology,2000, American Society of Plant Physiology, July 15-19, San Diego, California.

Carpita, N.C., Nabors, M.W., Ross, C.W., and Peteric, N.L. (1979). The growthphysics and water relations of red light induced germination in lettuce seeds: III.Changes in the osmotic and pressure potential in the embryonic axes of red andfar-red treated seeds. Planta 144: 217-224.

Carpita, N.C., Ross, C., and Nabors, M. (1979). The influence of plant growth regu-lators on the growth of the embryonic axes of red- and far-red-treated lettuceseeds. Planta 145: 511-516.

Casal, J.J. and Sánchez, R.A. (1998). Phytochromes and seed germination. SeedScience Research 8: 317-329

Casal, J.J., Sánchez, R.A., and Botto, F.J. (1998). Modes of action of phytochromes.Journal of Experimental Botany 49: 127-138.

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Chen, F. and Bradford, K.J. (2000). Expression of an expansin is associated with en-dosperm weakening during tomato seed germination. Plant Physiology 124:1265-1274.

Chen, F., Dahal, P., and Bradford, K.J. (2001). Two tomato expansins genes showdivergent expression and localization in embryos during seed development andgermination. Plant Physiology 127: 928-936.

Dahal, P., Nevins, D.J., and Bradford, K.J. (1996). Endosperm cell wall sugar com-position and sugars released during tomato seed germination. Plant Physiology111: 160.

Dahal, P., Nevins, D.J., and Bradford, K.J. (1997). Relationship of endo- -man-nananse activity and cell wall hydrolysis in tomato endosperm to germinationrates. Plant Physiology 113: 1243-1252.

de Miguel, L., Burgin, J., Casal, J.J., and Sánchez, R.A. (2000). Antagonistic actionof low-fluence and high-irradiance modes of response of phytochrome on germi-nation and -mannanase activity in Datura ferox seeds. Journal of ExperimentalBotany 51: 1127-1133.

de Miguel, L.C. and Sánchez, R.A. (1992). Phytochrome-induced germination, en-dosperm softening and embryo growth potential in Datura ferox seeds: Sensitiv-ity to low water potential and time to escape to FR reversal. Journal of Experi-mental Botany 43: 969-974.

Debeaujon, I. and Koornneef, M. (2000). Gibberellin requirement for Arabidospsisthaliana seed germination is determined both by testa characteristics and embry-onic ABA. Plant Physiology 122: 415-424.

Derkx, M.P.M. and Karssen, C.M. (1993). Effects of light and temperature on seeddormancy and gibberellin-stimulated germination in Arabidopsis thaliana—Studies with gibberellin-deficient and gibberellin-insensitive mutants. Physi-ologia Plantarum 89: 360-368.

Finkelstein, R.R., Gampala, S.S.L., and Rock, C.D. (2002). Abscisic acid signalingin seeds and seedlings. Plant Cell 14: S15-S45.

Grappin, P., Bouinot, D., Sotta, B., Miginiac, E, and Jullien, M. (2000). Control ofseed dormancy in Nicotiana plumbaginifolia: Post imbibition abscisic acid syn-thesis imposes dormancy maintenance. Planta 210: 279-285.

Groot, S.P.C. and Karssen, C.M. (1987). Gibberellins regulate seed germination intomato by endosperm weakening: A study with gibberellin-deficient mutants.Planta 172: 525-531.

Groot, S.P.C. and Karssen, C.M. (1992). Dormancy and germination of abscisicacid-deficient tomato seeds: Studies with the sitiens mutant. Plant Physiology99: 952-958.

Groot, S.P.C., Kieliszewska-Rockika, B., Vermeer, E., and Karssen, C.M. (1988).Gibberellin-induced hydrolysis of endosperm cell walls in gibberellin-deficienttomato seeds prior to radicle protrusion. Planta 174: 500-504.

Gwynn, D. and Schiebe, J. (1972). An action spectra for inhibition of lettuce seed.Planta 144: 121-124.

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Hartmann, K.M. (1966). A general hypothesis to interpret “high energy phenom-ena” of photomorphogenesis on the basis of phytochrome. Photochemistry andPhotobiology 5: 349-366.

Hennig, L., Stoddart, W.M., Dieterle, M., Whitelam, G.C., and Schäfer, E. (2001).Phytochrome E control light-induced germination of Arabidopsis. Plant Physi-ology 128: 194-200.

Hilhorst, H.W.M. (1995). A critical update on seed dormancy: I. Primary dor-mancy. Seed Science Research 5: 61-74.

Hilhorst, H.W.M. and Karssen, C. (1992). Seed dormancy and germination: Therole of abscisic acid and gibberellins and the importance of hormone mutants.Plant Growth Regulation 11: 225-238.

Insausti, P., Soriano, A., and Sánchez, R.A. (1995). Effects of flood-related factorson seed germination of Ambrosia tenuifolia. Oecologia 103: 127-132.

Jacobsen, S. and Olszewski, N. (1993). Mutation at the SPINDLY locus altergibberellin signal transduction. Plant Cell 5: 87-896.

Karssen, C. and Lacka, E. (1986). A revision of the hormone balance theory of seeddormancy: Studies on gibberellin and/or abscisic acid-deficient mutants ofArabidopsis thaliana. In M. Bopp (Ed.), Plant Growth Substances 1985 (pp. 315-323). Heidelberg, Germany: Springer-Verlag.

Koornneef, M., Bentsink, L., and Hilhorst, H. (2002). Seed dormancy and germina-tion. Current Opinion in Plant Biology 5: 33-36.

Koornneef, M. and van der Veen, J. (1980). Induction and analysis of gibberellinsensitive mutants in Arabidopsis thaliana (L.) Heynh. Theoretical Applied Ge-netics 58: 257-263.

Le Page-Degivry, M. and Garello, G. (1992). In situ abscisic acid synthesis: A re-quirement for induction of embryo dormancy in Helianthus annuus. Plant Phys-iology 98: 1386-1390.

Lee, S., Cheng, H., King, K., Wang, W., He, Y., Hussain, A., Lo, J., Harberd, N., andPeng, J. (2002). Gibberellin regulates Arabidopsis seed germination via RGL2, aGAI/RGA-like gene whose expression is up regulated following imbibition.Genes and Development 16: 646-658.

Leubner-Metzger, G., Fründt C., and Meins, F., Jr. (1996). Effects of gibberellins,darkness and osmotica on endosperm rupture and class I -1,3-glucanase induc-tion in tobacco seed germination. Planta 199: 282-288.

Leubner-Metzger, G., Fründt, C., Voegeli-Lange, R., and Meins, F., Jr. (1995).Class I -1,3-glucanases in the endosperm of tobacco during germination. PlantPhysiology 109: 751-759.

Malacoste, R., Tzanni, H., Jaques, R., and Rollin, P. (1972). The influence of bluelight on dark red germinating seeds of Nemophyla insignis. Planta 103: 24-34.

Mancinelli, A.L. (1980). The photoreceptors of the high irradiance responses ofplant photomorphogenesis. Photochemistry and Photobiology 32: 853-857.

Mandoli, D.F. and Briggs, W.R. (1981). Phytochrome control of two low-irradianceresponses in etiolated oat seedlings. Plant Physiology 67: 733-739.

Mella, R.A., Burgin, M.J., and Sánchez, R.A. (2004). Expansin gene expression inDatura ferox L. seeds is regulated by the low-fluence response, but not by thehigh-irradiance response, of phytochromes. Seed Science Research 14: 61-72.

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Mella, R., de Miguel, L., Garzarón, I., and Sánchez, R. (2002). Single seed studiesof germination inhibition by continuous far-red light in Datura ferox L. seeds.VII International Workshop on Seed Biology (pp. 113), Salamanca, Spain, May12-16.

Mella, R.A., Maldonado, S., and Sánchez, R.A. (1995). Phytochrome-inducedstructural changes and protein degradation prior to radicle protrusion in Daturaferox seeds. Canadian Journal of Botany 73: 1371-1378.

Ni, B.R. and Bradford, K.J. (1993). Germination and dormancy of abscisic acid- andgibberellin-deficient mutant tomato (Lycopersicon esculentum) seeds. PlantPhysiology. 101: 607-617.

Nonogaki, H., Gee, O., and Bradford, K. (2000). A germination specific endo- -mannanase gene is expressed in the micropylar endosperm cap of tomato seeds.Plant Physiology 123: 1235-1245.

Nonogaki, H. and Morohashi, Y. (1996). An endo- -mannanase develops exclu-sively in the micropylar endosperm of tomato seeds prior to radicle emergence.Plant Physiology 110: 555-559.

Nonogaki, H. and Morohashi, Y. (1999). Temporal and spatial patterns of endo- -mannanase expression in lettuce seeds. IV International Workshop on Seeds Bi-ology, Merida, Mexico, January.

Nonogaki, H., Nomaguchi, M., and Morohashi, Y. (1995). Endo- -mannanases inthe endosperm of germinated tomato seeds. Physiologia Plantarum 94: 328-334.

Nonogaki, H., Nomaguchi, M., and Morohashi, Y. (1998). Temporal and spatialpattern of the biochemical activation of the endosperm during and following im-bibition of tomato seeds. Physiologia Plantarum 102: 236.

Peng, J. and Harberd, N.P. (2002). The role of GA-mediated signaling in the controlof seed germination. Current Opinion in Plant Biology 5: 376-371.

Psaras, G. and Georghiu, K. (1983). Gibberellic acid-induced structural alterationsin the endosperm of germinating Latuca sativa L. achenes. Zeitschrift fürPfanzenphysiologie 112: 15-19.

Psaras, G., Georghiu, K., and Mitrakos, K. (1981). Red-light induced endospermpreparation for radicle protrusion of lettuce embryos. Botanical Gazette 142:13-18.

Sánchez, R.A. and de Miguel, L. (1985). The effect of red light, ABA and K+ on thegrowth rate of Datura ferox embryos and its relations with the photocontrol ofgermination. Botanical Gazette 146: 472-476.

Sánchez, R.A. and de Miguel, L. (1997). Phytochrome promotion of mannan-degrading enzyme activities in the micropylar endosperm of Datura ferox seedsrequires the presence of the embryo and gibberellin synthesis. Seed Science Re-search 7: 27-33.

Sánchez, R., de Miguel, L., Lima, C., and Lederkremer, R. (2002). Effect of low wa-ter potential on phytochrome-induced germination, endosperm softening andcell wall mannan degradation. Seed Science Research 12: 155-163.

Sánchez, R.A., Sunell, L., Labavitch, J., and Bonner, B.A. (1990). Changes in endo-sperm cell walls of two Datura species before radicle protrusion. Plant Physiol-ogy 93: 89-97.

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Schopfer, P. and Plachy, C. (1985). Control of seed germination by abscisic acid: II.Effect on embryo growth potential (minimum turgor pressure) and growth coef-ficient (cell wall extensibility) in Brassica napus L. Plant Physiology 77: 676-686.

Schopfer, P. and Plachy, C. (1993). Photoinhibition of radish (Raphanus sativus L.)seed germination: Control of growth potential by cell-wall yielding in the em-bryo. Plant, Cell and Environment 16: 223-229.

Scopel, A.L., Ballaré, C.L., and Sánchez, R.A. (1991). Induction of extreme lightsensitivity in buried weed seeds and its role in the perception of soil cultivations.Plant, Cell and Environment 14: 501-508.

Shichijo, C., Katada, K., Tanaka, O., and Hashimoto, T. (2001). PhytochromeA-mediated inhibition of seed germination in tomato. Planta 213: 764-769.

Shinomura, T., Nagatani, A., Hanzawa, H., Kubota, M., Watanabe, M., and Furuya,M. (1996). Action spectra for phytochrome A- and phytochrome B-specificphotoinduction of seed germination in Arabidopsis thaliana. Proceedings of theNational Academy of Sciences, USA 93: 8129-8133.

Steber, C. and McCourt, P. (2001). A role for brassinosteroids in germination inArabidopsis. Plant Physiology 125: 763-769.

Tillberg, E. and Björkman, P.-O. (1993). Effect of red and far-red irradiation onABA and IAA content in Pinus sylvestris L. seeds during the escape time periodfrom photocontrol. Plant Growth Regulation 13: 1-6.

Toorop, P.E., van Aelst, A.C., and Hilhorst, H.W.M. (1998) Endosperm cap weak-ening and endo- -mannanse activity during priming of tomato (Lycopersiconesculentum Mill cv. Money Maker) are initated upon crossing a threshold waterpotential. Seed Science Research 8: 483-491.

Toorop, P.E., van Aelst, A.C., and Hilhorst, H.W.M. (2000). The second step of thebiphasic endosperm cap weakening that mediates tomato (Lycopersicon escu-lentum) seed germination is under control of ABA. Journal of Experimenal Bot-any 51: 1371-1379.

Toyomasu, T., Kawaide, H., Mitsihashi, W., Inoue, Y., and Kamiya, Y. (1998).Phytochrome regulates gibberillin biosynthesis during germination of photo-blastic lettuce seeds. Plant Physiology 118: 1517-1523.

Toyomasu, T., Tsuji, H., Yamane, H., Nakayama, M., Yamaguchi, I., Murofushi,N., Takahashi, N., and Inoue, Y. (1993). Light effects on endogenous levels ofgibberellins in photoblastic lettuce seeds. Journal of Plant Growth Regulation.12: 85-90.

Vázquez-Yañez, C., Rojas-Aréchiga, M., Sánchez-Coronado, M.E., and Orozco-Segovia, A. (1996). Comparison of light-regulated seed germination in Ficusspp. and Cecropia obtusifolia: Ecological implications. Tree Physiology 16:871-875.

Vázquez-Yañez, C. and Smith, H. (1982). Phytochrome control of seed germinationin the tropical rain forest pioneer trees Cecropia obtusifolia and Piper auritumand its ecological significance. New Phytologist 92: 477-485.

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Watkins, J.T. and Cantliffe, D.J. (1983). Mechanical resistance of the seed coat andendosperm during germination of Capsicum annuum at low temperature. PlantPhysiology 72: 146-150.

Watkins, J.T., Cantliffe, D.J., Huber, D.J., and Nell, T. (1985). Gibberellic acidstimulated degradation of endosperm in pepper. Journal of the American Societyof Horticultural Science 110: 61-65.

Welbaum, G.E., Muthui, W.J., Wilson, J.H., Grayson, R.L., and Fell, R.D. (1995).Weakening of muskmelon perisperm envelope tissue during germination. Jour-nal of Experimental Botany 46: 391-400.

Wu, C., Leubner-Metzger, G., Meins, J.G., and Bradford. K.J. (2001). Class I -1,3-glucanase and chitinase are expressed in the micropylar endosperm of tomatoseeds prior to radicle emergence. Plant Physiology 126: 1299-1313.

Yamaguchi, S., Smith, M.W., Brown, R.G.S., Kamiya, Y., and Sun, T. (1998).Phytochrome regulation and differential expression of gibberillin 3 -hydroxy-lasegenes in germinating Arabidopsis seeds. Plant Cell 10: 2115-2126.

Yang, Y.Y., Yamaguchi, I., Takenowada, K., Suzuki, Y., and Murofushi, N. (1995).Metabolism and translocation of gibberellins in seedlings of Pharbitis nil: 1. Ef-fect of photoperiod on stem elongation and endogenous gibberellins in cotyle-dons and their phloem exudates. Plant Cell Physiology 36: 221-227.

Yoshioka, T., Endo, T., and Satoh, S. (1998). Restoration of seed germination atsupraoptimal temperatures by fluridone, an inhibitor of abscisic acid biosyn-thesis. Plant Cell Physiology 210: 307-312.

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Chapter 8

Modeling Changes in Dormancy in Weed Soil Seed BanksModeling Changes in Dormancyin Weed Soil Seed Banks: Implicationsfor the Prediction of Weed Emergence

Diego BatllaBetina Claudia Kruk

Roberto L. Benech-Arnold

INTRODUCTION

The seedling stage is a common target of many weed mechanical controland herbicide methods because of its high vulnerability (Fenner, 1987). Thesuccess of control methods targeted at weed seedlings depends upon reach-ing the highest number of individuals at this developmental stage. However,it is practically impossible to determine which proportion of a certain weedpopulation is being reached by a control method. Indeed, the number ofemerged seedlings can be counted, but we do not really know which frac-tion of the population they represent. Construction of weed seed germina-tion models that predict which proportion of the seed bank germinates at acertain time would be useful tools for determining the most suitable time forseedling control and, consequently, should result in a higher efficacy ofcontrols methods (Benech-Arnold and Sánchez, 1995). Although manymodels that successfully predict seed germination have been developed,one of the most important limitations for the formulation of such models inmany common weed species is the existence of dormancy. The lack of de-tailed research intending to understand and quantify how environmentalfactors regulate dormancy status in field situations probably prevented theelaboration of an adequate theoretical framework for the construction ofpredictive models addressing dormancy changes in weed seed bank popula-tions.

In the first section of this chapter we discuss the different environmentalfactors affecting dormancy in weed seed banks and present a general frame-work for classifying and understanding the effects of these factors on weed

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seed dormancy changes under field situations. The aim of this classificationis merely to facilitate the conceptualization of the whole system. The re-mainder of the chaper will discuss some attempts to model weed seed dor-mancy in relation to the effect of those environmental factors.

DORMANCY: DEFINITIONS AND CLASSIFICATION

Although many studies have been published concerning weed seed dor-mancy, the definition of dormancy is still a controversial subject. A newgeneral definition of dormancy was recently proposed by Benech-Arnoldand colleagues (2000, p. 106): “Dormancy is an internal condition of theseed that impedes its germination under otherwise adequate hydric, thermaland gaseous conditions.” This implies that once the impedance has been re-moved seed germination would proceed under a wide range of environmen-tal conditions (Benech-Arnold et al., 2000).

Karssen (1982) suggested that dormancy could be classified into pri-mary and secondary dormancy. Primary dormancy refers to the innate dor-mancy possessed by seeds when they are dispersed from the mother plant.Secondary dormancy refers to a dormant state that is induced in non-dormant seeds by unfavorable conditions for germination, or reinduced inonce-dormant seeds after a sufficiently low dormancy has been attained.Thus, it is by no means a classification referring to mechanisms or location,but one of timing of occurrence.

The release from primary dormancy followed by subsequent entranceinto secondary dormancy (whenever conditions are given for this entrance)may lead to dormancy cycling. Evidence for dormancy cycling has been ob-tained for seeds of many weed species, but it is not the only possibility. Forthe case of species that present dormancy cycling under natural field condi-tions, the transition into and out of dormancy may continue to cycle for sev-eral years before the seeds germinate, decay, or are otherwise lost from thesoil seed bank (Karssen, 1980/1981; Baskin and Baskin, 1985). In general,seeds are released from dormancy during the season preceding the periodwith favorable conditions for seedling development, whereas dormancy isinduced in the season preceding the period with harmful conditions forplant survival. For example, several summer annual species present a highdormancy level in autumn; during winter they undergo dormancy relief butdormancy increases again during summer. Winter annual species show thereverse dormancy pattern. Therefore, the patterns of dormancy are of highsurvival value to weed species determining germination under environmen-tal conditions that will ensure species growth and perpetuation (Karssen,1982).

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HOW IS DORMANCY LEVEL EXPRESSED?

Dormancy is not an all-or-nothing seed property; seed dormancy statuscan vary over a continuous dormancy degree scale between some point atwhich environmental conditions permissive for seed germination are nar-rowest and some point where environmental conditions permissive for seedgermination are widest. Vegis (1964) was the first to introduce this conceptof degrees of relative dormancy from the observation that as dormancy is re-leased, the temperature range permissive for germination widens until it ismaximal; in contrast, as dormancy is induced, the range of temperaturesover which germination can proceed narrows, until germination is no lon-ger possible at any temperature and full dormancy is reached. Clearly,Vegis’s view relates the degree of dormancy of a seed population to thewidth of the thermal range permissive for germination. Karssen (1982)agreed with that view and emphasized that seasonal periodicity in the fieldemergence of annual weeds is the combined result of seasonal periodicity infield temperatures and the width of the temperature range permissive forgermination. Germination in the field is therefore restricted to the periodwhen the field temperature and the temperature range over which germina-tion can proceed overlap.

The concept of base water potential for seed germination ( b) is fullydeveloped in Chapter 1. Many experimental data show that dormancy alle-viation could also be correlated with a decrease of the b of the seed popu-lation (i.e., more negative values), whereas dormancy induction could beassociated with an increase of the b of the seed population (i.e., less nega-tive or even positive values) (Dahal, Bradford, and Haigh, 1993; Ni andBradford, 1992, 1993; Bradford and Somasco, 1994). Based on these find-ings, Bradford (1995) proposed that changes in the dormancy level of seedpopulations could be associated with changes in the seed population b re-quired for germination. Thus, analogous to the thermal range permissive forgermination, the dormancy status of a seed population could also be evalu-ated by monitoring changes in the range of water potentials permissive forseed germination.

In many weed species, dormancy must be terminated by the effect oflight, nitrate, or fluctuating temperatures to allow the germination processto proceed. In those cases, changes in degree of dormancy comprise changesnot only in temperature requirements for germination (and eventually in

b), but also in sensitivity to the effect of dormancy terminating factors(Benech-Arnold et al., 2000).

Seedling emergence of a particular weed in the field occurs when envi-ronmental conditions are within the range of conditions permissive for seed

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germination (temperature range, water potential range, and requirement ofdormancy terminating factors stimuli). Since permissive conditions forseed germination change together with changes in dormancy level of seedpopulations, in order to construct dynamic models for predicting seedlingemergence we should understand the way in which the environment deter-mines changes in seed environmental requirements for germination as dor-mancy is released or enforced. To achieve this aim, it is necessary (1) toidentify the environmental factors involved in the modulation of dormancylevel of seed populations and those necessary for dormancy termination,and (2) to establish functional relationships between these factors and therates of these processes.

ENVIRONMENTAL FACTORS AFFECTING DORMANCYLEVEL OF SEED POPULATIONS

Dormancy cycles observed in some species are known to be regulatedmainly by temperature in temperate environments in which water is not sea-sonally restricted (Baskin and Baskin, 1977, 1984; Kruk and Benech-Arnold, 1998). For example, in some summer annual species dormancy re-lief is produced by low temperatures experienced during winter, and theirdormancy level is enhanced by high temperatures experienced during sum-mer (Bouwmeester and Karssen, 1992, 1993a). Several winter annual spe-cies show the reverse dormancy pattern. Hence, high temperatures duringsummer result in dormancy relief, and low temperatures during winter caninduce secondary dormancy (Baskin and Baskin, 1976; Karssen, 1982;Probert, 1992). As mentioned previously, these changes in dormancy levelcan be expressed through narrowing and widening of the temperature rangepermissive for germination. Thus, when soil temperature enters that per-missive range, germination in the field occurs (Figure 8.1). This range ischaracterized by two thermal parameters: (1) the mean lower limit tempera-ture (Tl50) and (2) the mean higher limit temperature (Th50). It should benoted that these parameters are conceptually different from base tempera-ture (Tb) and maximum temperature (Tm) (as described in Chapter 1). In-deed, while Tb and Tm are theoretical temperatures over and under whichthermal time ( ) is accumulated toward germination, Tl50 and Th50 repre-sent the temperatures under and over which dormancy is expressed for 50percent of the population. A main difference between these two kinds of pa-rameters is that Tb and Tm are regarded as unique for the whole population,whereas Tl and Th are normally distributed within the population (Washi-tani, 1987; Kruk and Benech-Arnold, 2000). The fact that each individualhas a different value of Tl and Th is consistent with the idea that dormancy

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level is different for each individual seed within the population. This con-cept will be further discussed in other sections of this chapter. Changes inthe dormancy level (or variations in the thermal range in which germinationcan occur) in summer species are characterized by an increase or decreaseof Tl (Baskin and Baskin, 1980). In contrast, in winter species changes indormancy level are characterized by fluctuations in Th (Figure 8.1).

Although a good deal of experimental data support a main role of soiltemperature as regulator of dormancy level in seed bank populations, someevidence indicates that the effect of temperature on dormancy release andinduction may be modulated by soil moisture (Adámoli, Goldberg, andSoriano, 1973; de Miguel and Soriano, 1974; Reisman-Berman, Kigel,and Rubin, 1991; Christensen, Meyer, and Allen, 1996; Bauer, Meyer, andAllen, 1998). Some interactions with soil moisture were detected in Poly-gonum aviculare L. seeds; dormancy release occurred most rapidly whenseeds were moist chilled at 4°C, but relief of dormancy was also possiblewith seeds dry stored at 4°C though at a much slower rate (Kruk and

0

10

20

30

40

Th

Tl

Tfield

Th

Tl

Tfield

O N D J F M A M J J A J J A S O N D J F M A M J J

Winter SpringWinter Spring Summer Autumn

Te

mp

era

ture

(ºC

)a) b)

FIGURE 8.1. Seasonal changes in the permissive germination thermal rangeand its relation with soil temperature dynamics. Solid lines indicate lower ( ) andhigher ( ) limit temperatures allowing germination. Broken lines show meandaily maximum temperature in the field. The shaded area represents the periodwhen field germination takes place due to overlapping of required and actualtemperature. (a) Strict summer annual; (b) facultative winter species. (Source:Adapted from Benech-Arnold et al., 2000.)

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Benech-Arnold, 1998). Batlla and Benech-Arnold (2000) also found thatP. aviculare seeds buried in the field under contrasting soil water contentconditions showed different annual pattern of changes in sensitivity to light,sensitivity to alternating temperatures, and the range of temperatures per-missive for germination. Other interactions have been also reported for thelight-requiring species Sisymbrium officinale L., for which a high sensitiv-ity to light stimuli, usually occurring in buried seeds at the end of the winter,is not acquired if the seeds have been permanently water imbibed and sub-jected to low winter temperatures (Hilhorst, Derkx, and Karssen, 1996). Onthe other hand, seeds composing the seed bank would be normally sub-jected to dehydration-hydration cycles, particularly in the upper layers ofthe soil. As rehydration of seeds previously imbibed and then dried wasfound to break dormancy in many weed species, Bouwmeester (1990) pro-posed that dehydration-hydration cycles can act as a dormancy-breakingenvironmental factor affecting buried seeds under field conditions.

In the field, induction of secondary dormancy can proceed at tempera-tures that are within the range suitable for germination. In those cases itmight result from inhibition of germination (i.e., germination-inhibitorywater potentials or inhibition of germination under leaf canopies), or from asituation in which factors that terminate dormancy are not met (i.e., loss ofsensitivity to light in light-requiring seeds held in darkness, loss of sensitiv-ity to fluctuating temperatures in seeds held at low thermal amplitudes)(Benech-Arnold et al., 2000). In any case, the process itself should involvethe narrowing of the range of suitable conditions for germination, ulti-mately leading to a state of relative or total dormancy, to be regarded as in-duction of secondary dormancy (Karssen, 1982).

FACTORS THAT TERMINATE DORMANCY

Once reaching a low dormancy level, several species require exposure tocertain environmental stimuli for the termination of dormancy. Fluctuatingtemperatures and light are predominantly naturally occurring environmen-tal factors that can complete exit from dormancy in many weed seeds(Benech-Arnold et al., 1990b; Scopel, Ballaré, and Sánchez, 1991; Ghersa,Benech-Arnold, and Martinez Ghersa, 1992), although other factors (i.e.,CO2, NO3

–, O2, and ethylene) can be involved in the termination of dor-mancy of buried seeds under field conditions (Benech-Arnold et al., 2000).An ecological interpretation of the requirement of light or fluctuating tem-peratures to complete exit from dormancy in certain weed species has beenrelated to the possibility of detecting canopy gaps as well as depth of burial(Holmes and Smith, 1977; Frankland, 1981; Thompson and Grime, 1983;

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Benech-Arnold et al., 1988; Deregibus et al., 1994; Batlla, Kruk, andBenech-Arnold, 2000). The requirement of fluctuating temperatures to ter-minate dormancy in some species has also been regarded as an effectivemechanism for distributing germination over a longer period of time (Benech-Arnold et al., 1990a,b).

Several characteristics of diurnal temperature cycles could be responsi-ble for its stimulatory activity (Roberts and Totterdell, 1981). Thermal am-plitude is of paramount importance; in Chenopodium album L., the dor-mancy breakage response can increase from an amplitude of as little as2.4°C up to about 15°C (Murdoch, Roberts, and Goedert, 1988). However,the response to a given amplitude is greater the higher the mean temperatureof the cycle (i.e., average of lower and upper temperature) up to an optimumof about 25°C (Murdoch, Roberts, and Goedert, 1988). In some cases, diur-nal temperature cycles with stimulatory characteristics tend to be additivein their effect. For example, in Sorghum halepense L. seeds, ten cycles withstimulatory characteristics release from dormancy twice the proportion ofthe population released with only five cycles (Benech-Arnold et al., 1990a).

In many weed species, dormancy is terminated when the hydrated seed isexposed to light, which is perceived through photoreceptors, particularlythose from the phytochrome family. Phytochromes have two mutuallyphotoconvertible forms: Pfr (considered the active form) with maximumabsorption at 730 nm and Pr with maximum absorption at 660 nm. Phyto-chromes are synthesized as Pr, and the proportion of the pigment population(P) in the active form (Pfr/P) in a particular tissue depends on the light envi-ronment seeds are exposed. Exposure of seeds to light with a high red (R) tofar-red (FR) ratio (R:FR) leads to larger Pfr/P determining, depending onseed dormancy level, breaking dormancy in many weed species. Phyto-chrome-mediated responses can be classified physiologically into three“action modes” (Kronenberg and Kendrick, 1986). Two of these actionmodes, the very low fluence response (VLFR) and the low fluence response(LFR), are characterized by the correlation between the intensity of the ef-fect and level of Pfr predicted to be established by the light environment.They differ in that extremely low levels of Pfr saturate VLFRs, while higherPfr levels are necessary to elicit LFRs (Casal and Sánchez, 1998). Highirradiance responses (HIRs) are the third action mode; they show no simplerelationship between Pfr levels and may involve additional components ofthe phytochrome system (Heim and Schäfer, 1982, 1984). Other particularcharacteristics of an HIR are that it has maximum activity at 710 to 720 nm(Hartman, 1966; Hendricks, Toole, and Borthwick, 1968; Mohr, 1972), andthe inhibitory effect of continuous FR can be observed, even in R-promotedseed, after the escape time is over (Frankland and Taylorson, 1983).

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Commonly crop or pasture leaf canopies reduce R:FR ratio perceived byweed seeds placed on the soil surface (Smith 1982; Pons 1992). Thischange in light quality would establish low Pfr/P, preventing weed seed ger-mination. The LFR and/or HIR mode of action can mediate this type of in-hibition by canopy presence (Deregibus et al., 1994; Batlla, Kruk, andBenech-Arnold, 2000). On the contrary, reductions in canopy density, forexample by grazing, mainly lead to increases in R:FR ratios which conse-quently raises Pfr/P seed level promoting germination by an LFR of manyweed seeds disposed on the soil surface (Deregibus et al., 1994; Insausti,Soriano, and Sánchez, 1995). An example of a VLFR is typically observedwhen soil is disturbed by agricultural practices. Seeds of several speciesmay acquire a very great sensitivity to light as dormancy is released througha period of burial in the soil (Scopel, Ballaré, and Sánchez, 1991). Theseseeds can respond to exposures of the order of sub-milliseconds of sunlightdetermining the germination of a large number of seeds from the seed bankwhen soil is being disturbed by tillage practices.

CONCEPTUALIZING THE SYSTEMWITH MODELING PURPOSES

Although real scenarios under field conditions are far more complicated,showing interactions of many kinds between relevant environmental factorsaffecting seed dormancy, the classification carried out so far is useful forunderstanding how the environment controls dormancy in weed soil seedbanks and, eventually, for developing simulation and predictive models.Thus, on the basis of the classification into two different kinds of environ-mental factors that affect dormancy as described in previous paragraphs,namely, (1) those that govern changes in the degree of dormancy of a seedpopulation (i.e., temperature and its interactions with soil hydric condi-tions) and (2) those that remove the ultimate constraints for seed germina-tion (i.e., mainly light and fluctuating temperatures), Benech-Arnold andcolleagues (2000) proposed the diagram shown in Figure 8.2. It illustratesthe conceptual framework derived from the definitions of the different fac-tors that affect dormancy in weed seed populations. It should be noted thatpassage along the whole flowchart is by no means the only possibility for aseed population. On the contrary, the chart aims to illustrate the differentpathways that a seed population could undergo. For example, a populationmight be dispersed with a low level of dormancy and might or might not re-quire limited stimuli for dormancy termination. In that case, the population

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would not experience the left side of the flowchart (unless induction of sec-ondary dormancy takes place) and may or may not bypass the zone of dor-mancy termination.

MODELING DORMANCY CHANGES IN WEED SEED BANKSAS AFFECTED BY THE ENVIRONMENT

Dormancy is probably the most important of a series of components andprocesses that affect seedling emergence in weed species (Forcella et al.,2000). Thus, in order to predict time and proportion of weed seed bankemergence, we should consider changes in dormancy as affected by envi-ronmental factors in the construction of our germination models. Abundantinformation has been published concerning dormancy in weed species asdescribed in the first part of this chapter, but few attempts have been madeto model seed dormancy changes as affected by the environment. Althougha vast review of existing models regarding seed dormancy is not intended in

DormancyEnforcement

Terminationof

Dormancy

DormancyAlleviation

Germination

HIGH

DORMANCY

LEVEL

LOW

DORMANCY

LEVELSEEDLINGS

Low sensitivity

to factors that

terminate dormancy

High sensitivity

to factors that

terminate dormancy

Narrow thermal

range permissive

for germination

Wide thermal

range permissive

for germination

High mean base

water potential

for germination [ (50)]Yb for germination [Yb (50)]

Lowmean basewater potential

Temperature

Moisture conditions?

Light:

Low R/FR (HIR)Skotodormancy

Hypoxia

Temperature

Moisture

conditions?

Fluct. Temp.

Light

CO : Low2

concentrations

NO3

Hypoxia

Ethylene

Temperature:

ThermalTime

HIGH

DORMANCY

LEVEL

Moistureconditions:Hydrotime

FIGURE 8.2. Flowchart representing changes in dormancy level and terminationof dormancy in seed populations and the factors that most likely affect each pro-cess (Source: Reprinted from Field Crops Research 67(2), R. L. Benech-Arnoldet al., Environmental control of dormancy in weed seed banks in soil, pp. 105-122, copyright 2000, with permission from Elsevier Science.)

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this section, some relevant attempts to model dormancy changes in weedspecies will be discussed.

The Role of Temperature

As mentioned earlier, soil temperature is one of the most important envi-ronmental factors controlling annual dormancy cycles of buried weed seedsin the field. Therefore, it is not surprising that almost all attempts to modeldormancy changes in weed seeds use temperature as the key factor drivingchanges in seed population dormancy status. Totterdell and Roberts (1979)were probably the first investigators to establish functional relationshipsbetween temperature and dormancy changes in weed seed populations.These authors hypothesized that temperature-dependent changes in dor-mancy of Rumex crispus L. and Rumex obtusifolius L. seeds result from twosimultaneous independent processes: (1) relief of primary dormancy and(2) induction of secondary dormancy. They suggest that relief of primarydormancy would occur only as a result of exposure of seeds to temperaturesunder a critical ceiling temperature for dormancy loss to occur. Accordingto these authors, such a process proceeds at a constant rate, independentlyfrom the actual temperature, as long as it is below that ceiling temperature.For the case of Rumex species the authors estimate a ceiling temperature fordormancy loss of 15°C. On the other hand, they suggest that induction ofsecondary dormancy would occur at all temperatures at a rate that wouldrise concomitantly with temperature.

Using Permissive Germination Thermal Rangefor Dormancy Modeling

Based on the hypothesis proposed by Totterdell and Roberts (1979) andthe concept of dormancy introduced by Vegis (1964), Bouwmeester andKarssen (1992) developed a descriptive simulation model that successfullypredicted changes in dormancy of buried seeds of the summer annualPolygonum persicaria L. as a function of soil thermal conditions. Themodel considers seed population dormancy status as a function of cold (C)and heat (H) unit sums. C is related to dormancy release, and H is related tosecondary dormancy induction. For simulation of seed population dor-mancy status, the value of C is raised by an arbitrary value of one unit foreach period of ten days during which the mean soil temperature has beenbelow the ceiling temperature for dormancy loss to occur, which forP. persicaria the authors determined to be 15°C. On the other hand, H is cal-culated by summing the mean soil temperature of each successive ten-day

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period. Thus, seed dormancy status depends on the balance between twoprocesses: dormancy alleviation, quantified by C, and dormancy induction,quantified by H. The authors observed that for seeds exhumed from the fieldat regular intervals, the germination percentage (Gt) obtained at three incu-bation temperatures could be described by a quadratic function of the ger-mination test temperature (Tg):

Gt = a Tg2 + b Tg + c (8.1)

where a, b, and c are functions of C, H, the presence or absence of nitrates inthe germination medium, and the mean soil temperature during 30 daysprior to seed exhumation. Using Equation 8.1 annual changes in seed ger-mination behavior in relation to soil temperature can be predicted. Themodel also allows the estimation of the width of the thermal range permis-sive for germination for seeds buried for different periods of time andexposed to a variable thermal environment. Narrowing or widening of thegermination permissive thermal range was the result of changes in the mini-mum temperature for germination of 50 percent of the seed population (pre-sumably analogous to the previously introduced Tl50). Therefore, germina-tion in the field is restricted to the period during which field temperatureand the germination permissive thermal range simulated by the model over-lap (Figure 8.3). Model performance showed good agreement between sim-ulated emergence timing for 50 percent of the seed population and observedtiming of germination of seeds disposed in petri dishes outdoors. The pres-ent model structure was also used by the same authors to predict timing offield emergence of Sisymbrium officinale L., Chenopodium album L., andSpergula arvensis L. (Bouwmeester, 1990; Bouwmeester and Karssen,1993a,b,c). However, results obtained with simulations showed that forthese weeds the model cannot give a description of dormancy relief as accu-rate as observed for P. persicaria, suggesting that temperature effects ondormancy changes would not be as simple as initially described by Totter-dell and Roberts (1979) for Rumex species.

Results obtained with other weed species also contrast with Totterdelland Roberts’s hypothesis (Pritchard, Tompsett, and Manger, 1996; Kebreaband Murdoch, 1999). For example, Batlla and Benech-Arnold (2003) ob-served no induction of secondary dormancy in P. aviculare seeds stratifiedfor 110 days under constant temperatures of 1.6, 7, and 12°C. Moreover, ex-humed seeds showed a progressive decrease in their dormancy level duringthe stratification period, verified by a widening of the thermal range permis-sive for germination. The rate of decrease in seed dormancy status wasshown to be negatively related to the stratification storage temperature. On

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the other hand, when seeds were stored for 12 days under a constant temper-ature of 22°C, secondary dormancy was rapidly induced. Exposed resultscontrast with Totterdell and Roberts’s hypothesis, suggesting that for P. avi-culare a threshold temperature for dormancy induction may exist and thatthe rate of dormancy release, under the ceiling temperature for this processto occur, would be dependent on the temperature at which seed after-ripen-ing takes place.

Using Base Water Potential for Dormancy Modeling

Although many attempts to quantify dormancy changes in weed seedpopulations were done by assessing changes in the permissive thermalrange for germination, Bradford (1996, 1997) proposed that dormancychanges can also be described and eventually predicted through changes inthe seed population base water potential ( b) used, in this case, as an indexof the seed population dormancy status. Working with the winter-annualweed Bromus tectorum L., Christensen, Meyer, and Allen, (1996) found

DD D JJ JJ JJ FF MM MM AA AA SS OO N J J JF M MA A S O NN

1987 1988 19891986

Tem

pera

ture

°C

–10

5

0

5

10

15

25

30

35

20

FIGURE 8.3. Simulated seasonal changes in the permissive thermal range forgermination of 50 percent of Polygonum persicaria seed population (maximumand minimum temperatures indicated by solid lines). Air temperature at 1.5 m isrepresented by the dashed line. Hatched areas represent the period of overlapbetween simulated permissive germination thermal range and actual tempera-ture. The arrows indicate the lapses at which germination of at least 50 percentof the population actually occurred in petri dishes placed outdoors. (Source:Oceologia, The dual role of temperature in the seasonal changes in dormancyand germination of seeds of Polygonum persicaria L., H. J. Bouwmeester andC. M. Karssen, Vol. 90, pp. 88-94, 1992, © Springer-Verlag. Reproduced withpermission.)

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that mean seed population base water potential [ b (50)] became more neg-ative as seeds after-ripened under dry conditions. Based on these findingsBauer, Meyer, and Allen, (1998) derived a simulation model to predictB. tectorum dormancy loss by dry after-ripening in the field for four seedpopulations corresponding to contrasting habitats. The model allowed b(50) to vary in relation to the accumulation of thermal time units (tempera-ture above a threshold temperature for dormancy loss to occur), while otherparameters of the hydrothermal time equation [the hydrothermal time con-stant ( HT), the standard deviation of b ( b), and the base temperature(Tb)] were held constant during after-ripening. Results showed that dor-mancy release was accompanied by a progressive decrease in b (50) andthat changes in b (50) can be described by a linear negative relationship tothermal after-ripening time accumulation (Figure 8.4):

b (50) = m [(Ts – Tl) (tar)] + b (8.2)

where b is the estimated initial value of b (50), m is the decrement in b(50) per unit of thermal time, Ts is the after-ripening storage temperature, Tlis the threshold temperature at or below which after-ripening does not oc-cur, and tar is the time required for full after-ripening.

Therefore, changes in b (50) account for changes in seed germinationtime-course curves due to variations in dormancy status or incubation tem-perature. Model performance was evaluated against b (50) values derivedfrom incubation of previously buried seeds retrieved from the field at regu-lar intervals. Hourly recorded seed-zone soil temperatures and estimatedseed-zone water potential were inputs for the model. Thus, b (50) de-creases as a function of after-ripening thermal-time accumulation, if esti-mated seed-zone water potential during that hour is considered low enoughfor after-ripening to occur (below approximately –4 MPa). The processcontinues until the b (50) value corresponding to fully after-ripened seedsis reached. Predictions of changes in b (50) were generally close to that es-timated for seeds retrieved from the field, suggesting that b (50) can beused as a reliable index to quantify changes in seed dormancy status.Derived model parameters used in Equation 8.2 were different for each pop-ulation, showing that model parameters should be adjusted for predictingdormancy changes in seed populations belonging to different habitats. An-other interesting feature of the model is that the effect of variable times andafter-ripening temperatures on seed dormancy status can be quantified as athermal-time phenomenon. A similar thermal-time approach was success-fully used by Pritchard, Tompsett, and Manger (1996) to quantify changes

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10/20°C incubation 20/30°C incubation

Mea

nba

sew

ater

pote

ntia

l (M

pa)

Estimated thermal time (degree-hours)

FIGURE 8.4. Dynamics of Bromus tectorum seed population b (50) deter-mined at two temperature regimes, plotted against thermal time accumulatedduring seeds dry after-ripening at different storage temperatures. Storage re-gimes are ( ) initial values, ( ) 10°C, ( ) 15°C, ( ) 20°C, ( ) 30°C, ( ) 40°C,and ( ) 50°C. Dotted horizontal lines correspond to b (50) for fully after-rip-ened seeds. (Source: M. C. Bauer, S. E. Meyer, and P. S. Allen, A simulationmodel to predict seed dormancy loss in the field for Bromus tectorum L., Journalof Experimental Botany, 1998, 49(324): 1235-1244, by permission of OxfordUniversity Press.)

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in the germination percentage of Aesculus hippocastanum L. seeds in rela-tion to after-ripening temperature. A more detailed review of the applica-tions of the hydrothermal time concept to quantify and model seed dor-mancy was recently done by Bradford (2002).

Modeling Dormancy As a Population-Based Phenomenon

Although existing models for simulation of dormancy changes underfield conditions can almost precisely predict the occurrence of the time win-dow for weed emergence, attempts to predict the proportion of seeds thatwill germinate at that time window with reasonable accuracy have, so far,been unsuccessful (Vleeshouwers, 1997; Murdoch, 1998). For achievingthis objective, we should be able to not only predict changes in mean popu-lation parameters that characterize seed dormancy status [namely, Tl50 andTh50 or b (50)], but also account for changes in their distribution within theseed population. Several reports on germination behavior of different spe-cies indicate that limit temperatures and water potentials demarcating thegermination range vary among individuals belonging to the same seed pop-ulation (Washitani, 1987, Bradford, 1996). Thus, if these parameters char-acterize the dormancy level of an individual seed, its distribution is a mea-sure of the spread of dormancy levels within the population. Quantificationand inclusion of this variation in dormancy models is essential for allowingthe estimation of germination percentage. The importance of accounting forthis variation to predict germination percentage of a seed population can beexemplified by considering two populations with the same Tl50 (i.e., 15°C),but different standard deviation of Tl ( Tl) (i.e., batch (A) Tl = 5; batch (B)

Tl = 1]: incubating seeds at 13.5°C will result in 35 percent of germinationin batch (A), while only 5 percent of the seed population will germinate inbatch (B) (Figure 8.5).

Recently, Batlla and Benech-Arnold (2003) developed a population-based threshold model for simulating P. aviculare seed dormancy loss in re-lation to stratification temperature. The model employs the mean lowerlimit temperature for germination (Tl50) as an index of seed population dor-mancy status, also accounting for changes in the distribution of Tl withinthe population ( Tl) in relation to seed population dormancy changes. Theauthors suggest that distribution of Tl is associated with variations in thedormancy status among seeds belonging to the same population. The ideathat dormancy is continuously distributed within individuals of a popula-tion, as referred to before, has been proposed frequently in the literature(Ransom, 1935; Probert, 1992; Bradford, 1996). Dynamic changes in these

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two parameters (Tl and Tl) as a function of a variable stratification thermalenvironment were predicted using thermal-time equations similar as thoseused by Bauer, Meyer, and Allen (1998) and Pritchard, Tompsett, andMager (1996). Accumulation of stratification thermal time (Stt) beganwhen temperature was below a threshold level for dormancy loss to occur(17ºC). Interestingly, although changes in Tl50 were linearly and negativelyrelated to accumulated Stt, the pattern of change in Tl was different de-pending on the temperature at which accumulation of Stt takes place. Thisimplies that under field situations changes in Tl would depend on the pre-vailing winter temperature at which dormancy release occurs. Model per-formance was evaluated against data of two unrelated experiments, show-ing acceptable prediction of timing and percentage of germination of seedsexhumed from field and controlled stratification conditions. A population-based threshold model structured to assess the combined effects of tempera-ture, water stress, and release from dormancy by cool-moist stratificationon the germination of seeds of Eucalyptus delegatensis R. T. Baker was alsodeveloped by Battaglia (1997). The model satisfactorily describes germina-tion of this species under a wide range of conditions, showing the ability ofpopulation-based threshold models to describe changes in dormancy andgermination behavior in response to environmental factors. The author pro-poses that the model structure can be easily modified in order to include ad-ditional factors and factor interactions controlling seed dormancy and ger-mination.

0 5 10 15 20 25 30 35

0

0.1

0.2

0.3

0.4

Temperature (°C)

Rela

tive

freq

uen

cy

inp

op

ula

tio

n

�Tl

= 5

35 %

0 5 10 15 20 25 30 35

Temperature (°C)

�Tl

= 1

5 %

Germinating seeds Germinating seeds

A B

FIGURE 8.5. Hypothesized normal distributions of Tl values in two seed popula-tions with the same Tl50 = 15°C (indicated by hatched arrows) but contrasting Tl( Tl A = 5; Tl B = 1). Striped areas represent the fraction of the seed populationthat would germinate if the two seed populations were incubated at 13.5°C (incu-bation temperature is indicated by solid arrows).

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Modeling Changes in Sensitivity to DormancyTerminating Factors

As pointed out previously, in many weed seed populations, additionalexposure to light, nitrate, or fluctuating temperatures may be required to al-low the germination process to proceed. For example, Sorghum halepenseL. seeds, a common summer weed in Argentinean pampas, present an abso-lute requirement of fluctuating temperatures for dormancy termination(Benech-Arnold et al., 1990b). Benech-Arnold and colleagues (1990a) de-veloped a dynamic model for prediction of S. halepense seed germinationin relation to soil temperature. The model basically states that two differentfractions can be identified within a S. halepense seed population in relationto their dormancy level: those seeds that must be stimulated by fluctuatingtemperatures to terminate dormancy and those that will not be releasedfrom dormancy after exposure to fluctuating temperatures. Based on thisclassification, the model calculates the proportion of the seed populationwhose dormancy is terminated after experiencing a certain number of cy-cles of fluctuating temperatures with stimulatory characteristics. To bestimulatory, a cycle must have a defined composition in terms of thermalamplitude and upper temperature. For example, for freshly dispersed seeds,effective cycles must have at least 15°C of thermal amplitude and 30°C ormore of upper temperature. Cycles with stimulatory characteristics have ad-ditive effects, each cycle releasing an additional proportion of the popula-tion from dormancy. The model assumes that cycle requirements for dor-mancy breakage can be satisfied even if those cycles are not met in acontinuous sequence. However, as stated previously, changes in degree ofdormancy comprise changes not only in temperature or water potential re-quirements for germination, but also in sensitivity to the effects of dor-mancy-terminating factors (Forcella et al., 2000). Thus, changes in the de-gree of dormancy in seeds that require fluctuating temperatures to terminatedormancy are likely to comprise changes in sensitivity to such fluctuations.To account for these changes, the model “looses” the thermal amplitude andupper temperature requirements for a cycle to produce a stimulatory effecton seeds that had after-ripened in the soil for a winter. Model performancewas successfully validated against independent field data, using eitherfreshly harvested or after-ripened seeds. Figure 8.6 shows the importance ofconsidering the dormancy terminating effect of fluctuating temperatureswhen modeling weed seedling emergence. Two models were run to simu-late seedling emergence under bare and shaded soil thermal conditions:(1) a simple thermal time model that ignores the seed requirements in termsof fluctuating temperatures, and (2) the previously-described model that

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considers the effect of fluctuating temperatures on seed dormancy. Thethermal-time model not only predicted anticipated seedling emergence inrelation to observed data, but also failed to distinguish between bare soiland shaded soil; clearly, seedling recruitment was much higher under baresoil because of the higher number of stimulatory cycles experienced by theseeds under this situation.

Although the model recognizes changes in seed sensitivity to the effectsof fluctuating temperature as seeds are released from dormancy, it does itonly discretely (i.e., freshly harvested seeds or seeds buried in the soil for awinter), and does not account for continuous dynamic changes in the re-sponse to alternating temperatures as seeds are released from or forced intodormancy. Moreover, changes in sensitivity to fluctuating temperaturesshould be different between years or locations, depending on soil tempera-tures experienced by the seeds during winter. Recently, Batlla, Verges, andBenech-Arnold (2003) showed that, for the summer annual P. aviculare,dormancy loss is indeed accompanied by changes in the sensitivity to fluc-tuating temperatures. These changes were characterized by a decrease inthe number of cycles required to achieve maximal germination responseand a progressive loss in the requirement of temperature fluctuations fordormancy breaking in further fractions of the seed population, as dormancy

FIGURE 8.6. Cumulative number of Sorghum halepense L. seedlings recordedin field plots under two different soil regimes, bare soil (open triangles) andshaded soil surface (open squares). The observed data are compared with datacorresponding to simulations done with a simple thermal-time model that ignoresseed requirements of fluctuating temperature for dormancy breaking (full cir-cles), and with a model that considers the effect of fluctuating temperature ondormancy breaking (full triangles and full squares).

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relief progresses. The authors relate seed response to the effect of cycledoses of fluctuating temperatures to accumulated Stt and observed thatseeds stratified at different temperatures which had accumulated equal val-ues of Stt showed similar response curves. Interestingly, as previouslyshown for the prediction of changes in Tl50 by the same authors (Batlla andBenech-Arnold, 2003), these preliminary results suggest that changes inseed sensitivity to cycle doses of fluctuating temperatures as a function ofstratification temperature could be also predicted using Stt.

Derkx and Karssen (1994) showed that changes in dormancy of Arabi-dopsis thaliana L. seeds buried in the field were associated with changes inthe sensitivity of the population to light stimuli. Sensitivity to light wasshown to increase when dormancy was alleviated and to decrease when dor-mancy was enforced. Modeling changes in light sensitivity as a result ofchanges in seed dormancy level would be useful for predicting the time atwhich seed maximum sensitivity to light is attained in the field. Accurateprediction of maximum sensitivity to light in relation to soil temperaturewould permit to plan soil disturbance by tillage practices in order to pro-duce the germination of an important fraction of the seed bank and, conse-quently, increase the efficacy of herbicide applications or cultural weedcontrol methods. Recently, Vleeshouwers and Bouwmeester (2001) devel-oped a mechanistic model for the simulation of dormancy changes in buriedlight-requiring weed seeds that is based on a physiological model concern-ing the action of phytochrome in the seed. Dormancy changes are driven byseasonal changes in soil temperature; this part of the model is based on re-lationships between temperature and changes in dormancy previously de-rived by Bouwmeester (1990) for C. album, S. arvensis, and P. persicariaseeds. In the model, the rate of dormancy release has a species-specific opti-mum temperature, ranging from 0 to 15°C, and decreases linearly at bothsides of this optimum, limited by a minimum and a maximum dormancy re-lease temperature. On the other hand, the rate of dormancy induction inC. album and P. persicaria increases linearly as soil temperature increasesover a minimum temperature for the process to occur. For S. arvensis therate of dormancy induction was characterized through an optimum, mini-mum, and maximum temperature. In contrast to the hypothesis of Totterdelland Roberts (1979), according to which dormancy induction and release aresimultaneous processes, in this model an internal switch determines whetherthe prevailing temperature has a dormancy-relieving or -inducing effect.Thus, in this model periods of dormancy release and induction are strictlyseparated depending on the prevailing temperature. The dormancy model iscoupled to a germination model that calculates germination percentages ofseed samples irradiated with red light and tested for germination at differenttemperatures. In general, annual dormancy changes are fairly well pre-

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dicted by the model for the three tested species. However, comparison withexperimental data shows that, as yet, the model is not accurate enough to beused in the prediction of field emergence patterns (Vleeshouwers andKropff, 2000).

Including Changes in Soil Water Content As a FactorThat Can Modulate Changes in Dormancy Status

Although models presented so far are basically based on the effect oftemperature on seed dormancy changes, changes in soil water content canaffect dormancy status in many weed seeds under field situations (Egley,1995). An example of the inclusion of soil water content as an environmen-tal factor affecting dormancy status of buried weed seeds in predictive mod-els was the previously described model for dry after-ripening of B. tectorumdeveloped by Bauer, Meyer, and Allen (1998). In B. tectorum seeds, dry af-ter-ripening occurs only when soil water content is below –4 MPa (Chris-tensen, Meyer, and Allen, 1996). The authors include this restriction for thedry after-ripening process to occur in the model; therefore, stratificationthermal time is accumulated only during hourly periods when the soil watercontent is below the threshold for dry after-ripening to proceed. Althoughweed seeds composing the seed bank are commonly subjected to soil watercontent fluctuations, models addressing seed dormancy changes rarely con-sider the effects of this factor. Future research to understand and quantifythe effect of soil water status on dormancy of weed seeds will be essentialfor accurately modeling weed emergence under real field situations.

CONCLUDING REMARKS

Although several attempts were made to model dormancy changes ofweed seed banks, further research is needed in order to successfully achievethis goal. Based on results obtained so far, important considerations for fur-ther research directions are as follows:

1. Although temperature has been shown to be the key factor regulatingdormancy changes of weed seed banks in the field, effects of other en-vironmental factors, such as changes in soil water status, nitrate con-tent, oxygen concentration, etc., should be considered under certainsituations. Experiments conducted to understand and quantify the ef-

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fects of environmental factors, other than temperature, on seed dor-mancy status changes would permit their inclusion in future simula-tion models addressing dormancy and germination.

2. Many environmental factors naturally occurring under field situa-tions, such as soil moisture content fluctuations, fluctuating tem-peratures, light, nitrates, etc., can affect seed dormancy status andconsequently weed emergence. This generates a difficult scenario formodeling dormancy changes due to multifactorial effects and interac-tions that should be considered in order to accurately predict dor-mancy and germination in real field situations. Including as many en-vironmental factors effects as possible (at least those consideredrelevant for the process) in our simulation models will lead to a betterprediction of weed emergence. For this purpose, strong quantitativerelationships between changes in the sensitivity to environmental fac-tors and changes in seed population dormancy status have to be de-rived.

3. Seed dormancy is a population-based phenomenon (Bradford, 2002).Thus, quantifying seed-to-seed variation in the response to environ-mental factors is a key feature for understanding and modeling seeddormancy (Murdoch, 1998). Population-based threshold models, suchas those proposed by Bradford (1996), Battaglia (1997), and Batllaand Benech-Arnold (2003), are encouraging attempts for modelingdormancy as a population phenomenon. In many cases, seed popula-tion responses to environmental factors can be characterized by amean population threshold response, which corresponds to 50 percentof the population, its standard deviation (dispersion around mean re-sponse), and an associated development-time constant (Battaglia,1997). Quantifying changes in these parameters for the response tonaturally occurring environmental factors, such as light, temperature,etc., in relation to seed dormancy changes would permit the integra-tion of seed population responses to the effect of many environmentalfactors in population-based threshold models.

4. The effect of temperature on seed dormancy status can be easily quan-tified using thermal-time equations, as done by Bauer, Meyer, andAllen (1998), Pritchard, Tompsett, and Manger (1996), and Batlla andBenech-Arnold (2003). This would permit referral of changes in theresponse to different environmental factors to a common thermal-time scale.

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REFERENCES

Adámoli, J.M., Goldberg, A.D., and Soriano, A. (1973). El desbloqueo de lassemillas de chamico (Datura ferox L.) enterradas en el suelo: Análisis de losfactores causales. Revista de Investigaciones Agropecuarias, Serie 2, 10: 209-222.

Baskin, J.M. and Baskin, C.C. (1976). High temperature requirement for after-ripening in seeds of winter annuals. New Phytologist 77: 619-624.

Baskin, J.M. and Baskin, C.C. (1977). Role of temperature in the germination ecol-ogy of three summer annual weeds. Oecologia 30: 377-382.

Baskin, J.M. and Baskin, C.C. (1980). Ecophysiology of secondary dormancy inseeds of Ambrosia artemisiifolia. Ecology 61: 475-480.

Baskin, J.M. and Baskin, C.C. (1984). Role of temperature in regulating timing ofgermination in soil seed reserves of Lamium purpureum (L.). Weed Research 30:341-349.

Baskin, J.M. and Baskin, C.C. (1985). The annual dormancy cycle in buried weedseeds: A continuum. BioScience 35: 392-398.

Batlla, D. and Benech-Arnold, R.L. (2000). Effects of soil water status and depth ofburial on dormancy changes of Polygonum aviculare L. seeds. In Abstracts ofthe Third International Weed Science Congress (p. 22). Corvallis, OR: Interna-tional Weed Science Society.

Batlla, D. and Benech-Arnold, R.L. (2003). A quantitative analysis of dormancyloss dynamics in Polygonum aviculare L. seeds: Development of a thermal timemodel based on changes in seed population thermal parameters. Seed ScienceResearch 13: 55-68.

Batlla, D., Kruk, B., and Benech-Arnold, R.L. (2000). Very early detection of can-opy presence by seeds through perception of subtle modifications in R:FR sig-nals. Functional Ecology 14: 195-202.

Batlla, D., Verges, V., and Benech-Arnold, R.L. (2003). A quantitative analysis ofseed responses to cycle-doses of fluctuating temperatures in relation to dor-mancy: Development of a thermal time model for Polygonum aviculare L. seeds.Seed Science Research 13: 197-207.

Battaglia, M. (1997). Seed germination model for Eucalyptus delegatensis prove-nances germinating under conditions of variable temperature and water poten-tial. Australian Journal of Plant Physiology 24: 69-79.

Bauer, M.C., Meyer, S.E., and Allen P.S. (1998). A simulation model to predict seeddormancy loss in the field for Bromus tectorum L. Journal of Experimental Bot-any 49: 1235-1244.

Benech-Arnold, R.L., Ghersa, C.M., Sánchez, R.A., and García Fernandez, A.(1988). The role of fluctuating temperatures in the germination and establish-ment of Sorghum halepense (L.) Pers. regulation of germination under leaf cano-pies. Functional Ecology 2: 311-318.

Benech-Arnold, R.L, Ghersa, C.M., Sánchez, R.A., and Insausti, P. (1990a). Amathematical model to predict Sorghum halepense germination in relation tosoil temperature. Weed Research 30: 81-89.

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Benech-Arnold, R.L., Ghersa, C.M., Sánchez, R.A., and Insausti, P. (1990b). Tem-perature effects on dormancy release and germination rate in Sorghum halepense(L.) Pers. seeds: A quantitative analysis. Weed Research 30: 91-99.

Benech-Arnold, R.L. and Sánchez, R.A. (1995). Modeling weed seed germination.In Kigel, J. and Galili, G. (Eds.), Seed Development and Germination (pp. 545-566). New York: Marcel Dekker.

Benech-Arnold, R.L., Sánchez, R.A., Forcella, F., Kruk, B.C., and Ghersa, C.M.(2000). Environmental control of dormancy in weed seed banks in soil. FieldCrops Research 67: 105-122.

Bouwmeester, H.J. (1990). The effect of environmental conditions on the seasonaldormancy pattern and germination of weed seeds. PhD thesis. Wageningen Ag-ricultural University, The Netherlands.

Bouwmeester, H.J. and Karssen, C.M. (1992). The dual role of temperature in theregulation of the seasonal changes in dormancy and germination of seeds ofPolygonum persicaria L. Oecologia 90: 88-94.

Bouwmeester, H.J. and Karssen, C.M. (1993a). Annual changes in dormancy andgermination in seeds of Sisymbrium officinale (L.) Scop. New Phytologist 124:179-191.

Bouwmeester, H.J. and Karssen, C.M. (1993b). The effect of environmental condi-tions on the dormancy pattern of Spergula arvensis. Canadian Journal of Botany71: 64-73.

Bouwmeester, H.J. and Karssen, C.M. (1993c). Seasonal periodicity in germinationof seeds of Chenopodium album L. Annals of Botany 72: 463-473.

Bradford, K.J. (1995). Water relations in seed germination. In Kigel, J. and Galili,G. (Eds.), Seed Development and Germination (pp. 351-395). New York: Mar-cel Dekker.

Bradford, K.J. (1996). Population-based models describing seed dormancy behav-iour: Implications for experimental design and interpretation. In Lang, G.A.(Ed.), Plant Dormancy: Physiology, Biochemistry, and Molecular Biology (pp. 313-339). Wallingford, UK: CAB International.

Bradford, K.J. (1997). The hydrotime concept in seed germination and dormancy.In Ellis, R.H., Black, M., Murdoch, A.J., and Hong, T.D. (Eds.), Basic and Ap-plied Aspects of Seed Biology (pp. 349-360). Dordrecht, the Netherlands: Klu-wer Academic Publishers.

Bradford, K.J. (2002). Applications of hydrothermal time to quantifying and model-ing seed germination and dormancy. Weed Science 50: 248-260.

Bradford, K.J. and Somasco, O.A. (1994). Water relations of lettuce seed thermo-inhibition: I. Priming and endosperm effects on base water potential. Seed Sci-ence Research 4: 1-10.

Casal, J.J. and Sánchez, R.A. (1998). Phytochromes and seed germination. SeedScience Research 8: 317-329.

Christensen, M., Meyer, S.E., and Allen, P.S. (1996). A hydrothermal time model ofseed after-ripening in Bromus tectorum L. Seed Science Research 6: 147-153.

Dahal P., Bradford, K.J., and Haigh, A.M. (1993). The concept of hydrothermaltime in seed germination and priming. In Côme, D. and Corinbeau, F. (Eds.),

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Fourth International Workshop on Seeds: Basic and Applied Aspects of Seed Bi-ology, Volume 3 (pp. 1009-1014). Paris: ASFIS.

de Miguel, L.C. and Soriano, A. (1974). The breakage of dormancy in Datura feroxseeds as an effect of water absorption. Weed Research 14: 265-270.

Deregibus, V.A., Casal, J.J., Jacobo, E.J., Gibson, D., Kauffman, M., and Rodri-guez, A.M. (1994). Evidence that heavy grazing may promote the germination ofLolium multiflorum seeds via phytochrome-mediated perception of high red/far-red ratios. Functional Ecology 8: 536-542.

Derkx, M.P.M. and Karssen, C.M. (1994). Are seasonal dormancy patterns inArabidopsis thaliana regulated by changes in seed sensitivity to light, nitrate andgibberellin? Annals of Botany 73: 129-136.

Egley G. (1995). Seed germination in soil. In Kigel, J. and Galili, A. (Eds.), SeedDevelopment and Germination (pp. 529-543). New York: Marcel Dekker.

Fenner, M. (1987). Seedlings. New Phytologist 106: 35-47.Forcella, F., Benech-Arnold, R.L., Sánchez, R.A., and Ghersa, C.M. (2000). Model-

ing seedling emergence. Field Crops Research 67: 123-139.Frankland, B. (1981). Germination in shade. In Smith, H. (Ed.), Plants and the Day-

light Spectrum (pp. 187-204). London: Academic Press.Frankland, B. and Taylorson, R.B. (1983). Light control of seed germination. In

Shropshire, W. and Mohr, H. (Eds.), Encyclopedia of Plant Physiology, Volume16A (pp. 428-456). New York: Springer Verlag.

Ghersa, C.M., Benech-Arnold, R.L., and Martinez Ghersa, M.A. (1992). The role offluctuating temperatures in germination and establishment of Sorghum hale-pense (L.) Pers: II. Regulation of germination at increasing depths. FunctionalEcology 6: 460-468.

Hartmann, K.M. (1966). A general hypothesis to interpret “high-energy phenom-ena” of photomorphogenesis on the basis of phytochrome. Photochemistry andPhotobiology 5: 349-366.

Heim, B. and Schäfer, E. (1982). Light-controlled inhibition of hypocotyl growth inSinapis alba L. seedlings. Planta 154: 150-155.

Heim, B. and Schäfer, E. (1984). The effect of red and far-red light in the highirradiance reaction of phytochrome (hypocotyl growth in dark-grown Sinapisalba L.). Plant, Cell and Environment 7: 39-44.

Hendricks, S.B., Toole, V.K., and Borthwick, H.A. (1968). Opposing action of lightin seed germination of Poa pratensis and Amaranthus arenicola. Plant Physiol-ogy 43: 2023-2028.

Hilhorst, H.W.M., Derkx, M.P.M., and Karssen, C.M. (1996). An integrating modelfor seed dormancy cycling: Characterization of reversible sensitivity. In Lang,G.A. (Ed.), Plant Dormancy: Physiology, Biochemistry, and Molecular Biology.Wallingford, UK: CAB International.

Holmes, M.G. and Smith, H. (1977). The function of phytochrome in the natural en-vironment: II. The influence of vegetation canopies on the spectral energy distri-bution of natural daylight. Photochemistry and Photobiology 25: 539-545.

Insausti, P., Soriano, A., and Sánchez, R.A. (1995). Effects of flood-influenced fac-tors on seed germination of Ambrosia tenuifolia. Oecologia 103: 127-132.

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Karssen C.M. (1980/1981). Patterns of change in dormancy during burial of seedsin soil. Israel Journal of Botany 29: 65-73.

Karssen, C.M. (1982). Seasonal patterns of dormancy in weed seeds. In Khan, A.A.(Ed.), The Physiology and Biochemistry of Seed Development, Dormancy andGermination (pp. 243-270). Amsterdam: Elsevier.

Kebreab, E. and Murdoch, A.J. (1999). A quantitative model for loss of primarydormancy and induction of secondary dormancy in imbibed seeds of Orobanchespp. Journal of Experimental Botany 50: 211-219.

Kronenberg, G.H.M and Kendrik, R.E. (1986). The physiology of action. In Kendrik,R.E. and Kronenberg, G.H.M. (Eds.), Photomorphogenesis in Plants (pp. 99-114). Dordrecht, the Netherlands: Marthinus Nijhoff/Dr. W. Junk Publishers.

Kruk, B.C. and Benech-Arnold, R. (1998). Functional and quantitative analysis ofseed thermal responses in prostate knotweed (Polygonum aviculare) and com-mon purslane (Portulaca oleracea). Weed Science 46: 83-90.

Kruk, B.C. and Benech-Arnold, R.L. (2000). Evaluation of dormancy and germina-tion responses to temperature in Carduus acanthoides and Anagallis arvensis us-ing a screening system, and relationship with field-observed emergence patterns.Seed Science Research 10: 77-88.

Mohr, H. (1972). Lectures on Photomorphogenesis. Berlin: Springer Verlag.Murdoch, A.J. (1998). Dormancy cycles of weed seeds in soil. Aspects of Applied

Biology 51: 119-126.Murdoch, A.J., Roberts, E.H., and Goedert, C.O. (1988). A model for germination

responses to alternating temperatures. Annals of Botany 63: 97-111.Ni, B.R. and Bradford, K.J. (1992). Quantitative models characterizing seed germi-

nation responses to abscisic acid and osmoticum. Plant Physiology 98: 1057-1068.

Ni, B.R. and Bradford, K.J. (1993). Germination and dormancy of abscisic acid- andgibberellin-deficient mutant tomato (Lycopersicon esculentum) seeds. PlantPhysiology 101: 607-617.

Pons, T.L. (1992). Seed response to light. In Fenner, M. (Ed.), The Ecology of Re-generation in Plant Communities (pp. 259-284). Melksham, UK: CAB Interna-tional.

Pritchard, H.W., Tompsett, P.B., and Manger, K.R. (1996). Development of a ther-mal time model for the quantification of dormancy loss in Aesculus hippo-castanum seeds. Seed Science Research 6: 127-135.

Probert, R.J. (1992). The role of temperature in germination ecophysiology. InFenner, M. (Ed.), The Ecology of Regeneration in Plant Communities (pp. 285-325). Melksham, UK: CAB International.

Ransom, E.R. (1935). The inter-relations of catalase, respiration, after-ripening, andgermination in some dormant seeds of the Polygonaceae. American Journal ofBotany 22: 815-825.

Reisman-Berman, O., Kigel, J., and Rubin, B. (1991). Dormancy patterns in buriedseed of Datura ferox L. Canadian Journal of Botany 69: 173-179.

Roberts, E.H. and Totterdell, S. (1981). Seed dormancy in Rumex species in re-sponse to environmental factors. Plant, Cell and Environment 4: 97-106.

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Scopel, A.L., Ballaré, C.L. and Sánchez, R.A. (1991). Induction of extreme lightsensitivity in buried weed seeds and its role in the perception of soil cultivations.Plant, Cell and Environment 14: 501-508.

Smith, H. (1982). Light quality, photoperception, and plant strategy. Annual Reviewof Plant Physiology 33: 481-518.

Thompson, K. and Grime, J. (1983). A comparative study of germination responsesto diurnally fluctuating temperatures. Journal of Applied Ecology 20: 141-156.

Totterdell, S. and Roberts, E.H. (1979). Effects of low temperatures on the loss ofinnate dormancy and the development of induced dormancy in seeds of Rumexobtusifolius L. and Rumex crispus L. Plant, Cell and Environment 2: 131-137.

Vegis, A. (1964). Dormancy in higher plants. Annual Review of Plant Physiology15: 185-224.

Vleeshouwers, L.M. (1997). Modeling weed emergence patterns. PhD thesis, Agri-cultural University, Wageningen, The Netherlands.

Vleeshouwers, L.M. and Bouwmeester, H.J. (2001). A simulation model for sea-sonal changes in dormancy and germination of seeds. Seed Science Research 11:77-92.

Vleeshouwers, L.M. and Kropff, M.J. (2000). Modeling field emergence patterns inarable weeds. New Phytologist 148: 445-457.

Washitani, I. (1987). A convenient screening test system and a model for thermalgermination responses of wild plant seeds: Behaviour of model and real seed inthe system. Plant, Cell and Environment 10: 587-598.

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SECTION III:SEED LONGEVITY AND STORAGE

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Chapter 9

Orthodox Seed Deterioration and Its RepairOrthodox Seed Deterioration and Its Repair

Miller B. McDonald

INTRODUCTION

Seed deterioration can be defined as deteriorative changes occurringwith time that increase the seed’s vulnerability to external challenges anddecrease the ability of the seed to survive. Three general observations canbe made about seed deterioration. First, seed deterioration is an undesirableattribute of agriculture. Annual losses of revenue from seed/grain productsdue to deterioration can be as much as 25 percent of the harvested crop. Thisvalue would be in the billions of U.S. dollars (McDonald and Nelson,1986). An understanding of seed deterioration, therefore, provides a tem-plate for improved crop production as well as increased agricultural profits.Second, the physiology of seed deterioration is a separate event from seeddevelopment and/or germination. Thus, the knowledge gained from under-standing these events likely does not apply to what occurs during deteriora-tion. Third, seed deterioration is cumulative. As seed aging increases, seedperformance is increasingly compromised. With these tenets in mind, whatcauses seeds to die? An understanding of this process might begin with anunderstanding of seed evolution—a topic seldom discussed.

THE FIRST SEED

What was the first seed like? Since no humans were present at the timethe first seed was formed, it is difficult to answer this question with cer-tainty. However, trying to understand that first seed might help in a quest toincrease our knowledge of seed aging. In general, seeds are divided into twocategories based on their storage characteristics: recalcitrant and orthodox(although gradations may exist—see Pammenter and Berjak, 2000). Recal-

Salaries and research support were provided by state and federal funds appropriatedto the Ohio Agricultural Research and Development Center, Ohio State University.

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citrant seeds are desiccation intolerant (cannot be dried below approxi-mately 40 percent seed moisture content without damage) and are typicallycharacterized as large seeds with small embryos from tropical trees andshrubs (Chin and Roberts, 1980). Orthodox seeds, in contrast, are desicca-tion tolerant (can be dried to 5 percent seed moisture content without dam-age), often manifest some type of dormancy, and are characteristic of mostagriculturally important crops found worldwide. Orthodox seeds representmost of the seeds found in the world and are among the most agriculturallyimportant species. As a result of their common occurrence, it is tempting tospeculate that the first seed possessed orthodox behavior.

To determine whether the first seed was recalcitrant or orthodox, it is im-portant to remember the agricultural definition of a seed which emphasizesthat the propagule must be a reproductive unit. Thus, the first seed was oneproduced on the parent plant and later dispersed followed by germination,successfully regenerating the species. It is generally agreed that plant lifefirst originated in the tropics, an area where seasons are consistent and tem-peratures conducive for maximum plant growth. If plants first appearedthere, seeds could be shed at any time from the plant and, presuming ade-quate moisture, immediately resume growth without the need for dormancyor a quiescent period (Garwood, 1989; Vazquez-Yanes and Orozco-Segovia,1993). Recalcitrant seeds, in contrast, do not possess dormancy but, instead,must continue their development and progress toward germination (Berjaket al., 1990). Recalcitrant seeds are not desiccation tolerant and, if dried,will die. Although this trait led to successful plant reproduction, it made ag-ricultural planning difficult because seed life span was short. However, assuccessful plants expanded their range beyond the tropics, they encoun-tered differing seasons that required seed adaptations for survival. These in-cluded the ability to dry down so that respiration and physiology associatedwith growth were reduced during unfavorable seasonal climes as well as theimposition of dormancy. Therefore, desiccation tolerance and dormancymay be acquired traits. The ability of seeds to reduce their metabolism fol-lowing maturation, however, was a significant advantage for successful ag-riculture. For the first time, humans were able to harvest and store seeds forlong durations without loss in seed quality. This permitted shipment ofseeds to other locations as well as planting of seeds in subsequent seasonsfollowing long-term storage. The imposition of dormancy mechanisms that“sensed” the environment continues to be an agricultural challenge becausethe depth of dormancy likely varies from seed to seed in a seed lot. As a re-sult, both desiccation tolerance and dormancy have been adaptive advan-tages that contributed to the prevalence of orthodox seeds around the world.

Therefore, it appears that the first seed was a recalcitrant seed, a case ad-vocated by Pammenter and Berjak (2000). It was characterized by high

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moisture, high respiration, short life span, and an inability to dry downwithout seed damage. Orthodox seeds likely followed recalcitrant seeds,with the significant advantage that they developed desiccation-tolerantmechanisms which reduced respiration and ceased active embryo growth.Simultaneously, orthodox seeds increased their life span well beyond recal-citrant seeds. Why is that so, and what are the physiological mechanismsthat govern and regulate orthodox seed deterioration? The purpose of thischapter is to describe the processes that cause orthodox seed deteriorationand the physiological mechanisms that permit these seeds to survive long-term storage. From such knowledge, we gain insights into approaches forenhancing seed storage potential and improving seed quality.

SEED DETERIORATION

Seed deterioration is inexorable, and the best that can be done is to con-trol its rate. Many factors contribute to seed deterioration. These include ge-netics, mechanical damage, relative humidity and temperature of the stor-age environment, seed moisture content, presence of microflora, seedmaturity, etc. Of these, relative humidity and temperature are the two mostimportant. Relative humidity is important because it directly influences themoisture content of seeds in storage as they come to equilibrium with theamount of gaseous water surrounding them. Temperature is important be-cause it (1) determines the amount of moisture the air can hold (higher tem-peratures holding more water than lower temperatures) and (2) enhancesthe rate of deteriorative reactions occurring in seeds as temperature in-creases. These relationships are so important that Harrington (1972) identi-fied the following two rules of thumb describing seed deterioration:

Rule 1: Each 1 percent reduction in seed moisture content doubles thelife of the seed.

Rule 2: Each 5°C reduction in seed temperature doubles the life of theseed.

Harrington (1972) recognized that some qualifications to these ruleswere needed for them to be applied successfully. First, rule one does not ap-ply above 14 or below 5 percent seed moisture content. Seeds stored atmoisture contents above 14 percent begin to exhibit increased respiration,heating, and fungal invasion which destroy seed viability more rapidly thanindicated by the moisture content rule. Below 5 percent seed moisture, abreakdown of membrane structure hastens seed deterioration (probablya consequence of reorientation of hydrophyllic membranes due to the lossof the water molecules necessary to retain their structural configuration).

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For the second rule, for temperatures below 0°C the rule may not apply be-cause many biochemical reactions associated with seed deterioration do notoccur and further reductions in temperature have only a moderate effect inextending seed longevity. Finally, it should not be forgotten that these twofactors, seed moisture content and temperature, interact. This was capturedin another equation suggested by Harrington (1972) in which the sum of thetemperature in degrees Farenheit and the percentage relative humidityshould not exceed 100. From this equation, one can see that as the tempera-ture of the storage environment increases, the relative humidity must de-crease. The influence of seed moisture content, temperature, and orthodoxseed deterioration were demonstrated in hypothetical deterioration curvespresented in Figure 9.1 (Ellis, Osei-Bonsu, and Roberts, 1982).

This complex milieu of interacting factors makes the study of seed dete-rioration and its underlying physiology difficult. It is beyond the purview ofthis chapter to consider each of these factors, and the reader is encouragedto examine a book (Priestley, 1986) as well as a comprehensive chapter(Copeland and McDonald, 2001) and reviews (Halmer and Bewley, 1984;McDonald, 1985; McDonald and Nelson, 1986; Smith and Berjak, 1995;McDonald, 1999) on the subject. This chapter will consider orthodox seeddeterioration from a physiological perspective. Starting with a high-qualityseed under optimum storage conditions, what happens to the seed as itsquality is reduced?

Seed Deterioration Is Not Uniform

A general assumption is that seed deterioration occurs uniformlythroughout a seed, but a seed is a composite of tissues that differ in theirchemistry and proximity to the external environment. Thus, it should not beassumed that seed deterioration occurs uniformly throughout the seed. Per-haps the best example that this does not occur comes from the use of thetetrazolium chloride (TZ) test which causes living tissues in a seed to turnred (Association of Official Seed Analysts [AOSA], 2000; Society of Com-mercial Seed Technologists [SCST], 2001). The challenge to the seed re-searcher/analyst is to decipher how important the living (or dead) tissues areto successful seedling establishment. When studies have been conducted onseeds using controlled natural and artificial aging conditions, differences inthe deterioration of seed tissues have been observed. For example, in wheatseeds, deterioration begins with the root tip and progressively moves up-ward through the radicle, scutellum, and ultimately the leaves and coleop-tile (Das and Sen-Mandi, 1988, 1992). Similar findings have been reportedin maize in which root tip cells are the first to be damaged (Berjak, Dini, andGevers, 1986) which causes the rate of radicle extension to be lower thancoleoptile extension following aging (Bingham, Harris, and MacDonald,

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FIGURE 9.1.Theoretical distribution curves for soybean.Curves were computedfor various constant storage conditions: (1) different temperatures at 12 percentmoisture content with initial germinability 98 percent, (b) different moisture con-tents at 20oC with initial germinability 98 percent, and (c) different initial germin-abilities (98, 80, 60, and 40 percent) at 12 percent moisture content and 20oC.(Source: R. H. Ellis, K. Osei-Bonsu, and E. H. Roberts, The influence of geno-type, temperature, and moisture on seed longevity in chickpea, cowpea, andsoya bean, Annals of Botany, 1982, 50: 69-82, by permission of Oxford Univer-sity Press.)

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1994). Similarly, in dicot seeds such as soybean, root growth is more sensi-tive to accelerated aging than shoot growth (Hahalis and Smith, 1997) andthe embryonic axis more sensitive to deterioration than the cotyledons(Chauhan, 1985; Seneratna, Gusse, and McKersie, 1988; Tarquis and Brad-ford, 1992). These findings demonstrate that the embryonic axis is moreprone to aging in monocot and dicot orthodox seeds, and, of the axis struc-tures, the radicle axis is more sensitive to deterioration than the shoot axis.

Why this is so is not known, but at least two studies on the sequence ofwater uptake in soybean and maize seeds may provide an indication. Mc-Donald, Vertucci, and Roos (1988) showed that the soybean axis hydratedmore rapidly than the cotyledons. This was attributed to the presence of aradicle pocket in the seed coat that possessed large hourglass cells with a lowmatric potential for water (Figure 9.2). This external radicle pocket is in in-timate contact with the radicle and ensures that water absorbed during imbi-bition is attracted preferentially to the radicle axis. Similarly, McDonald,Sullivan, and Lauer (1994) described the uptake of water in a maize seed be-ginning with hydration in the radicle followed by the scutellum and then theshoot axis and coleoptile (Figure 9.3). They attributed this route to the openpores present in the remnants of the funiculus or collapsed black layerwhich afforded rapid water penetration into the seed. Although these resultswere for free-flowing water, it is likely that water present in the gaseous phaseof air would also be attracted by the same matric forces present in the seedcoat. This would result in higher water content in the embryo (and radicle)than in storage reserves (and other embryonic structures) which could selec-tively facilitate the events that cause seed deterioration in certain seed parts.

Physiology of Seed Deterioration

Our understanding of the events that cause seed deterioration remains in-complete. McDonald (1999) identified at least six reasons why it is difficultto critically evaluate seed deterioration studies:

1. The physiological processes governing seed deterioration vary. Forexample, short-term deterioration in the field is likely a differentphysiological event than long-term deterioration in storage.

2. Seed researchers use different methods to study seed deterioration.They can precisely control short-term seed deterioration under hightemperature, high relative humidity accelerated aging conditions, butis this process physiologically equivalent to the conditions occurringin natural, long-term storage conditions?

3. The rate of seed deterioration is influenced by confounding environ-mental and biological factors such as growth of storage fungi that cre-ate their own biological niche.

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4. Seed treatments influence seed deterioration, and, when applied, theirimpact on seed quality must be recognized.

5. Most seed deterioration studies examine whole seeds. As empha-sized, seed deterioration is not uniform within a seed and any study ofseed deterioration should begin with an understanding of where seeddeterioration occurs first.

6. Most seed deterioration studies report effects on a seed lot, but seeddeterioration is an individual event occurring in a population of seedscomposing the seed lot. Studies using bulk seeds are inappropriate.

FIGURE 9.2. Scanning electron micrograph showing the radicle pocket. (A)Sterographic micrograph of the concave seed coat surface with embryo removeddemonstrating the radicle pocket; (B) Cross section of the seed illustrating theradicle pocket enveloping the radicle of the embryonic axis; (C) magnified view ofthe radicle and radicle pocket; (D) cross section of the radicle pocket and radicle.Abbreviations: SC = seed coat, RP = radicle pocket, P = plumule, E = epicotyl,H = hypocotyl, R = radicle, HI = hilum, and C = cotyledon.Bars represent 1.0 mm.(Source: M. B. McDonald, C. W. Vertucci, and E. E. Roos, 1988, Soybean seedimbibition: Water absorption of seed parts, Crop Science, 28: 993-998. Repro-duced with permission from the Crop Science Society of America.)

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MECHANISMS OF ORTHODOX SEED DETERIORATION

Our quest to better understand orthodox seed deterioration has led to a va-riety of proposals. (Excellent and detailed considerations of these have beenprovided elsewhere and will not be the focus of this chapter; see Smith andBerjak, 1995; McDonald, 1999). These include changes in the following:

• Enzyme activities: Most of these studies search for markers of germi-nation such as increases in amylase activity or changes in free radicalscavenging enzymes such as superoxide dismutase, catalase, per-oxidase, and others.

• Protein or amino acid content: The consensus is that overall proteincontent declines while amino acid content increases with seed aging.

• Nucleic acids: A trend of decreased DNA synthesis and increasedDNA degradation has been reported. It is widely believed that degra-

FIGURE 9.3. Staining of the maize seed embryo with nitroblue tetrazolium chlo-ride at various intervals of soaking. Top left to right, 0, 3, and 6 h; bottom left toright, 15, 24, and 48 h. (Source: M. B. McDonald, J. Sullivan, and M. J. Lauer,1994, The pathway of water uptake in maize (Zea mays L.) seeds, Seed Scienceand Technology 22:79-90. Reproduced with permission.)

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dation of DNA would lead to faulty translation and transcription ofenzymes necessary for germination.

• Membrane permeability: Increased membrane permeability associ-ated with increasing seed deterioration has been consistently ob-served and is the foundation for the success of the conductivity test asa measure of seed quality.

FREE RADICAL PRODUCTION

Each of these general findings represent the result, not the cause, of seeddeterioration. As evidence mounts, the leading candidate causing seed dete-rioration increasingly appears to be free radical production. Free radicalproduction, primarily initiated by oxygen, has been related to the peroxi-dation of lipids and other essential compounds found in cells. This causes ahost of undesirable events including decreased lipid content, reduced respi-ratory competence, and increased evolution of volatile compounds such asaldehydes (Wilson and McDonald, 1986b).

Free Radicals—What Are They and Why Are They Important?

All atoms that make up molecules contain orbitals that occupy zero, one,or two electrons. An unpaired electron in an orbital carries more energythan each electron of a pair in an orbital. A molecule that possesses any un-paired electrons is called a free radical. Some free radicals are composed ofonly two atoms (O2

–) while others can be as large as protein or DNA mole-cules. Why is the free radical important in biological systems? The ener-getic “lonely electron” (1) can detach from its host atom or molecule andmove to another atom or molecule or (2) can pull another electron (whichmay not have been lonely) from another atom or molecule. The most com-mon free radical reaction is when one free radical and one non-free radicaltransfer one electron between them, leaving the free radical as a non-freeradical, while the non-free radical is now a free radical. This initiates achain of similar reactions which cause substantial damage in the intervalthat the reactions are occurring. Thus, free radicals can react with one an-other and with non-free radicals to change the structure and function ofother atoms and molecules. If these are proteins (enzymes), lipids (mem-branes), or nucleic acids (DNA), normal biological function is compro-mised and deterioration increased. The positive association of free radicalswith animal aging has recently been reviewed (Beckman and Ames, 1998).What still remains uncertain is their role in orthodox seed aging.

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FREE RADICALS AND THEIR EFFECTS ON LIPIDS

Lipid peroxidation begins with the generation of a free radical (an atomor a molecule with an unpaired electron) either by autoxidation or enzymat-ically by oxidative enzymes such as lipoxygenase present in many seeds.Various forms of free radicals have been observed or detected in living tis-sue, each with a differing capability for cell damage (Gille and Joenje,1991; Larson, 1997).

Superoxide anion (O2 ). Superoxide anion is produced by autoxidationof hydroquinones, leukoflavins, and thiols as well as enzymatically byflavoprotein dehydrogenases such as mitochondrial NADH dehydrogenase.

Hydrogen peroxide (H2O2). Hydrogen peroxide is produced by the spon-taneous or enzyme-catalyzed dismutation of O2 or by two-electron reduc-tion of O2. Flavoenzymes such as monoamine oxidase present on the outermitochondrial membrane of virtually all cells are probably the most impor-tant contributors to intracellular generation of H2O2. These enzymes whichnormally use O2 as a substrate catalyze two-electron transfer reactions thatproduce H2O2.

Hydroxyl radicals ( OH). Hydroxyl radicals are formed from O2 andH2O2 in the presence of iron which catalyzes the reaction. In cells, iron canbe bound to compounds such as adenosine triphosphate (ATP), guanidintriphosphate (GTP), and citrate, thereby forming a more soluble iron-che-late complex. OH is by far the most reactive oxygen radical, and it reactsalmost immediately with any molecule at the site where it is generated. Themain mechanism of toxicity of H2O2 and O2 may be their ability to com-bine to form OH.

Singlet oxygen (1O2). During lipid peroxidation, 1O2 can be generated inthe termination step:

LOO + LOO LO + LOH + 1O2

and in the reaction with triplet carboxyls formed during lipid peroxidation:

RO* + O2 LO + 1O2

Singlet oxygen combines with DNA bases causing genetic damage.

HOW DO FREE RADICALS CAUSE LIPID PEROXIDATION?

The mechanism of lipid peroxidation is often initiated by oxygen aroundunsaturated or polyunsaturated fatty acids such as oleic and linoleic acids

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found commonly in seed membranes and storage oils. The result is the re-lease of a free radical, often hydrogen (H ), from a methylene group of thefatty acid adjacent to a double bond. In other cases, the free radical hydro-gen may combine with other free radicals from carboxyl groups (ROOH)leaving a peroxy-free radical (ROO ). Once these free radicals are initiated,they continue to propagate other free radicals that ultimately combine, ter-minating the destructive reactions. In their wake, they create profound dam-age to membranes and changes in oil quality. As a result, long-chain fattyacids are broken into smaller and smaller compounds, some of these beingreleased as volatile hydrocarbons (Wilson and McDonald, 1986a; Esashi,Kamataki, and Zhang, 1997). The final consequence is loss of membranestructure, leakiness, and an inability to complete normal metabolism.

WHAT IS THE INFLUENCE OF SEED MOISTURE CONTENTON FREE RADICAL ASSAULT?

Lipid peroxidation occurs in all cells, but in fully imbibed cells, wateracts as a buffer between the autoxidatively generated free radicals and thetarget macromolecules, thereby reducing damage. Thus, as seed moisturecontent is lowered, autoxidation is more common and is accelerated by hightemperatures and increased oxygen concentrations. Lipid autoxidation maybe the primary cause of seed deterioration at moisture contents below 6 per-cent. Above 14 percent moisture content, lipid peroxidation may again bestimulated by the activity of hydrolytic oxidative enzymes such as lipoxy-genase, becoming more active with increasing water content. Between 6and 14 percent moisture content, lipid peroxidation is likely at a minimumbecause sufficient water is available to serve as a buffer against autoxi-datively generated free radical attack, but not enough water is present to ac-tivate lipoxygenase-mediated free radical production.

Lipoxygenases may contribute to cell degradation by modifying cellmembrane composition. In higher plants, two major pathways involvinglipoxygenase activity have been described for the metabolism of fatty acidhydroperoxides (Figure 9.4, Loiseau et al., 2001). One pathway producestraumatic acid, a compound that may be involved in plant cell wound re-sponse (Zimmerman and Coudron, 1979) and volatile C6-aldehydes andC6-alcohols shown to be correlated with seed deterioration (Wilson andMcDonald, 1986a). The other pathway produces jasmonic acid, a moleculethat may play a regulatory role in plant cells (Staswick, 1992; Sembdnerand Parthier, 1993). Lipoxygenases have been identified and associatedwith almost every subcellular body in plants (Losieau et al., 2001), so it islikely that they have important regulatory roles in development. This may

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include the deterioration of hydrated seeds through free radical production.For example, Zacheo and colleagues (1998) found increased lipoxygenaseactivity at high relative humidity (80 percent) and temperature (20oC) dur-ing natural aging of almond seeds. Other correlative studies implicatinglipoxygenases have been identified (McDonald, 1999). A direct study of theimportance of lipoxygenases during orthodox seed deterioration employingmutants was reported by Suzuki and colleagues (1996, 1999). They foundthat a rice mutant deficient in lipoxygenase-3 had fewer peroxidative prod-ucts and fewer volatile compounds during seed aging compared to the wildtype.

Thus, the mechanism of lipid peroxidation may be different under long-term aging (autoxidation) compared to accelerated aging (e.g., lipoxy-

Linolenic acid

LIPOXYGENASES

9-HPOT

or

13-HPOT

ALLENE OXIDESYNTHASE

HYDROPEROXIDELYASE

Allene oxide Hexenal 9-oxo-nonanoic acid3-Z,6Z-nonadienal

12-oxo-9-Z-dodecenoic acid

-ketols-ketols

Jasmonic acidTraumatic acid

FIGURE 9.4. Overview of the lipoxgenase pathway. 9-HPOT, 9(S)-hydroperoxy-trans-10,cis-12,cis-15-octadecatrienoic acid; 1-HPOT, 1(S)-hydroperoxy-cis-9,trans-11,cis-15-octadecatrienoic acid. (Source: From Loiseau et al., 2001. Re-printed by permission of CABI Publishing, Wallingford, Oxon, UK.)

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genase) conditions. This is consistent with the proposals by Wilson and Mc-Donald (1986b) and Smith and Berjak (1995) that seeds are exposed to sep-arate lipid peroxidative events during storage and during imbibition. Itshould be noted that oxygen is deleterious to seed storage based on this pro-posal, which is consistent with the success of hermetic seed storage andlipid peroxidation as a cause of membrane integrity loss.

DO FREE RADICALS ATTACK ONLY LIPIDS?

Free radicals attack compounds other than lipids. Changes in proteinstructure of seeds have been observed and attributed to free radicals (Mc-Donald, 1999). Soluble proteins may be attacked by different classes of oxi-dants (and be protected by different classes of antioxidants) than membraneproteins. The most reactive amino acids susceptible to oxidative damageappear to be cysteine, histidine, tryptophan, methionine, and phenylalanine,usually in that order (Larson, 1997).

Free radicals are also suspected of assault on chromosomal DNA. Poten-tial targets for oxidative damage in the DNA chain include the purine andpyrimidine bases as well as the deoxyribose sugar moieties (Larson, 1997).Specific damage to the bases may leave the strand intact, but modificationof sugar residues can also lead to strand breakage. This may explain the in-creased propensity for genetic mutations as seeds age. Many of these muta-tions are first detected as chromosomal aberrations that delay the onset ofmitosis necessary for cell division and germination.

WHY SUSPECT FREE RADICAL ATTACKON MITOCHONDRIA?

Three reasons exist to believe that free radical attack on mitochondriamay be a prime cause of seed deterioration. First, mitochondria are the siteof aerobic respiration. Thus, they are the prime “sink” for oxygen, some ofwhich can leak from the membranes during respiration to create free radi-cals. Second, mitochondria are indispensable to normal cell function. Theyuse oxygen and substrates to generate energy. Third, an important manifes-tation of seed deterioration is reduced seedling growth, perhaps a conse-quence of less efficient mitochondrial function.

Mitochondria contain an inner membrane encased in another outer mem-brane, and both membranes differ in many important ways. The inner mem-brane is intricately folded (structures called cristae) and has a much greatersurface area than the outer membrane. The cristae are also the site of elec-

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tron transport where lonely electrons can leak and cause damage to the ex-tensive membrane surface, thereby compromising essential energy produc-tion necessary for germination. The space enclosed by the inner membraneis called the mitochondrial matrix. This matrix is high in protein concentra-tion, containing many enzymes as well as their cofactors critical for oxida-tive phosphorylation. The matrix also contains a small amount of DNA(mtDNA) and ribosomes for decoding the DNA. The outer membrane is notfolded and has large holes in it that permit the passage of many large pro-teins.

Of these compounds and structures, mtDNA is the most critical for main-taining normal cell function, and a review of its structure and function inplants has been provided (Hanson and Otto, 1992). To better understandthis important role, it should be noted that mtDNA differs from nuclearDNA in two important ways. First, when a cell divides, both nuclear andmtDNA are separately replicated. Mitochondria can also divide in an activecell, requiring the creation of a new copy of mtDNA; mtDNA is importantfor the production of new mitochondria in rapidly dividing and physiologi-cally active cells such as those that occur during germination. Second, theenzymes encoded by mtDNA are absolutely essential for oxidative phos-phorylation. Thus, maintenance of mtDNA is vital for actively respiringcells, the cells responsible for seedling growth. As a result, any challengesto mtDNA would surely disrupt normal cellular growth and division.

Since it is now clear that mtDNA and mitochondria are essential formaintenance of cells during dry storage and growth of cells during germina-tion, an essential question is whether mtDNA or nuclear DNA are moreprone to free radical attack. Studies have now documented that mtDNA suf-fers more spontaneous changes in its DNA sequence compared to nuclearDNA in animal cells which results in the production of incorrect or trun-cated proteins (DeGrey, 1999). This greater susceptibility is attributed tothe following:

• mtDNA being more exposed to free radical attack than nuclear DNA:Mitochondria are the principal site of oxygen utilization which re-sults in a greater level of free radical production.

• mtDNA being “naked”: Nuclear DNA is protected by special pro-teins called histones that must be degraded by free radicals prior tonuclear DNA exposure. mtDNA is not surrounded by these protec-tive structures.

• The repair of nuclear DNA is more successful than mtDNA: Fewerrepair enzymes exist around mtDNA.

• mtDNA being circular and containing a high level of short sequenceswhich appear twice, some distance apart: If the circular mtDNA

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wraps around itself to form a figure 8, the two identical sequencesmay end up next to each other and a “crossover” may occur at whichthe strands come apart and join each other. This causes the circularDNA to possess two circles, each with only a subset of the necessarygenetic material. In addition, one of these circles will be without thenecessary D-loop, a small portion of the DNA that contains no genesbut is essential for initiating replication of the molecule. Thus, thecircle without the D-loop will never be replicated and will eventuallybe lost during division, with the final result being the deletion of criti-cal mtDNA information.

Oxidative damage is an important contributor to mutations in mtDNA.Various types of deletion mutations have been reported to increase with ag-ing (Kang et al., 1998). Accumulation of specific mutations in somatic cellswith aging may be due to either mutations occurring at particular sites (e.g.,hot spots) or randomly throughout the genome. In humans, these mutationshave been associated with specific mitochondrial diseases (Kang et al.,1998). In plants, early studies with plant mtDNA rearrangements haveshown clear associations with abnormal growth mutants, cytoplasmic malesterility (Newton and Gabay-Laughman, 1998), as well as abnormal pollendevelopment (Conley and Hanson, 1995) and protein formation (Lu et al.,1996). Whether these same changes occur in seeds has not been deter-mined. However, with the advent of the polymerase chain reaction (PCR), ithas become possible to extract low levels of mtDNA and amplify it for thedetermination of scissions possibly caused by free radical attack. In addi-tion, a technique called long PCR now allows the identification of differ-ent mtDNA deletions present in the whole mtDNA (Barnes, 1994) in con-trast to the original technique in which only a small proportion of sequencevariants were identified (Cheng, Higuchi, and Stoneking, 1994; Reynierand Mathiery, 1995). Using these PCR refinements, it has been demon-strated that point mutations can occur in mtDNA in animal tissues and accu-mulate with age (Munscher, Muller-Hocker, and Kadenbach, 1993; Kaden-bach, Munscher, and Frank, 1995).

How Are Free Radicals Produced in Mitochondria?

Mitochondria are the major source of reactive oxygen species (ROS)(Kang et al., 1998). Under normal physiological conditions, more than 1percent of the oxygen consumed by cells is converted to ROS. In animalsystems, this amounts to about 107 ROS molecules/mitochondrion/day. Mi-tochondrial respiration accounts for 90 percent of the cellular oxygen con-sumed, and the respiratory chain in mitochondria is principally responsible

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for the production of ROS (Kang et al., 1998). Free radicals are easilyformed during oxidative phosphorylation where the consumed oxygen isturned into water by the addition of four protons and four electrons. Theprotons and electrons reach their targets by different routes. The electronsare carried one by one along a chain of molecules (cytochromes, etc.). Eachtime this is done, the electron carrier is turned into a free radical. If they canpass their free radical onto the next carrier, no harm is done. However, this isnot a perfect system and sometimes the electrons escape at some stage in thechain. These loose electrons can form potentially toxic free radicals. Themajority of free radicals during oxidative phosphorylation are accepted byone omnipresent, molecular oxygen (O2). The resulting molecule is calledsuperoxide with a chemical formula of O2

–. The negative charge indicatesthat it has one more electron than proton, making it an anion, and the “ ” in-dicates it is a free radical.

Although superoxide is a free radical, it is one that does not contribute tosignificant cellular damage. Instead, superoxide is converted (usually bysuperoxide dismutase) into hydrogen peroxide (H2O2) that can accept anelectron from Fe2+ (or Cu+ ) and, in so doing, splits in two to form HO andwater. HO is vastly more reactive than superoxide and will readily initiatelipid peroxidation in the mitochondrial cristae.

In animals, the mitochondrial free radical theory of aging has become theleading candidate to explain cell aging (DeGrey, 1999). In particular, be-cause of free radical attack, the integrity of mtDNA becomes increasinglydamaged with age. For example, the tissues that exhibited the greatest lev-els of mtDNA damage were those that utilized the most energy per unit vol-ume and/or generated the most reactive molecules. In other words, highmetabolic rate shortens life span. Thus, because orthodox seeds are low inmoisture content and their metabolic rates are consequently low, they areable to survive for longer durations.

Peroxidation proceeds as a chain reaction so the products of peroxi-dation in an intact mitochondrion will be concentrated around the occa-sional point at which a reaction was initiated. These locally high levels ofmembrane damage constitute pinpricks in the membrane through whichprotons flow rapidly.

HOW ARE SEEDS PROTECTEDAGAINST FREE RADICAL ATTACK?

Seeds contain a complex system of antioxidant defenses to protectagainst the harmful consequences of activated oxygen species. At leastthree defenses in seeds protect against free radical attack.

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The first is an array of enzymes to neutralize activated oxygen species.Although these are unlikely to operate in dry seeds, their activity would bevital during imbibition. Specific enzymes exist that detoxify O2

–, H2O2,and organic peroxides. No enzymes have yet been found that detoxify OHor 1O2. Examples (Gille and Joenje, 1991) include the following.

Superoxide dismutase (SOD). Superoxide dismutase enzymes catalyzethe dismutation reaction:

2 O2 + 2H+ H2O2 + O2

SOD enzymes have been found in the cellular cytoplasm and matrix spaceof mitochondria.

Catalase (CAT). Catalase catalyzes the decomposition of hydrogen per-oxide to oxygen and water:

2 H2O2 2 H2O + O2

Catalase subunits are formed in the cytoplasm, and synthesis of the enzymeis completed in the peroxisome. Catalase is absent in the mitochondrial ma-trix of most cells.

Glutathione peroxidase (GP). Glutathione peroxidase catalyzes the re-moval of H2O2 and lipid peroxides:

LOOH + 2 GSH LOH + H2O + GSSG

Reduced glutathione (GSH) is then regenerated from the oxidized state(GSSG) by glutathione reductase, a reduction that consumes NADPH.

The second protective approach includes nonenyzmatic compounds thatreact with activated oxygen species and thereby block the propagation offree radical chain reactions. These include the following.

Glutathione (GSH). Glutathione is a water-soluble antioxidant found inthe cytoplasm which reacts with O2 , OH, and 1O2:

O2 + H+ + GSH GS + H2O2OH + GSH GS + H2O1O2 + GSH GSox (cysteic acid)

The glutathione radical (GS ) is considered relatively stable and causes lit-tle damage.

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Vitamin E (tocopherol). Vitamin E or tocopherol nonenzymatically re-duces polyunsaturated lipid peroxide free radicals:

Vit E + LOO Vit E + LOOH

Vitamin E also readily reacts with O2 and 1O2 and is a water-insolublecompound found in the lipid domains of membranes.

Vitamin C (ascorbic acid). Vitamin C is a water-soluble compound capa-ble of reacting with free radicals (R ) and O2 and OH. Vitamin C is alsothought to regenerate vitamin E by accepting the activated electron fromVit E .

The third type of defense is enzymes that specifically fix damage createdby free radicals. These include DNA repair enzymes that involve a combi-nation of base excision, nucleotide excision, or DNA mismatch repair activ-ity. These function in the following way (Rasmussen and Singh, 1998).

Base excision repair pathway. In the nucleus, the first step in the base ex-cision repair process involves the removal of damaged bases by a damage-specific DNA glycolase. Removal of the damaged base by glycolase createsan apurinic or apyrimidinic site in the DNA. In the next step, AP-endo-nulcease cleaves the phosphodiester bond, forming a nucleotide gap. Thegap is then filled and sealed by DNA polymerase and DNA ligase, respec-tively.

Nulecotide excision repair pathway. Oxidative lesions are removed byhydrolyzing phosphodiester bonds on both sides of the lesion. Two excisionmechanisms accomplish this removal: the endonuclease-exonuclease andthe excision nuclease mechanisms. A concerted action of at least 16 poly-peptides is involved in the repair.

DNA mismatch repair pathway. Oxidative damage to mtDNA can lead tomisincorporation of nucleotides during replication of mitochondria. In thenucleus, DNA mismatch repair corrects these types of mutations in duplexDNA during DNA replication.

Based on these findings, various approaches to protect orthodox seedsagainst antioxidant free radical scavengers exist. For example, one toco-pherol molecule may afford antioxidant protection to several thousand fattyacid molecules (Bewley, 1986). Soybean seeds have a lower tocopherolcontent following aging, suggesting that tocopherol is consumed and pro-tects the seed against free radical damage (Seneratna, Gusse, and McKersie,1988). Superoxide dismutase increases with accelerated aging in pigeonpeaseeds (Kalpana and Madhava Rao, 1994). Other enzymes, such as gluta-thione reductase, are antioxidants on the one hand but sources of free radi-cals on the other, so it is difficult to determine their protective qualities

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(DeVos, Kraak, and Bino, 1994). However, glutathione is an efficient anti-oxidant and has such a role in aged sunflower (DePaula et al., 1996) and wa-termelon (Hsu and Sung, 1997) seeds. Thus, the addition of antioxidantsmight afford seeds protection against free radical attack. For example, theaddition of 0.04 M ferrous sulfate reduced lipid peroxidation and increasedthe quenching of free radicals in soybean axes during the first hour of imbi-bition (Hailstones and Smith, 1991). Pretreatment of seeds with compoundssuch as dikegulac-sodium, ascorbic acid, cinnamic acid, and -tocopherolprior to accelerated and natural aging improved seed vigor and seed storageof rice (Bhattacharjee and Bhattacharyya, 1989), maize and mustard (Deyand Mukherjee, 1988), sunflower (Bhattacharjee and Gupta, 1985), Frenchbean, pea, lentil, and millet (Chhetri, Rai, and Bhattacharjee, 1993), andjute (Bhattacharjee, Chowdhury, and Choudhuri, 1986; Chowdhury andChoudhuri, 1994).

RAFFINOSE OLIGOSACCHARIDESAND THEIR PROTECTIVE ROLE

Raffinose oligosaccharides (RFOs) have been implicated as importantcomponents of cell membranes that maintain membrane integrity duringdrying and storage of orthodox seeds (Crowe, Crowe, and Hoekstra, 1989;Hoekstra, Crowe, and Crowe, 1992; Oliver, Crowe, and Crowe, 1998;Peterbauer and Richter, 2001). Sugars are also involved in maintaining thethree-dimensional structure of proteins that prevent their unfolding and de-naturation due to loss of associated water during seed dry down (Crowe,Hoekstra, and Crowe, 1992; Wolkers et al., 1998). RFOs also appear to beinvolved with sucrose in the formation of glasses in dry seeds (Burke, 1986),which are highly viscous solids that retard molecular diffusion and slowdeteriorative reactions (Buitink et al., 2000). Glass formation in orthodoxseeds as they dry down during seed maturation may protect both lipids andproteins against free radical attack. All of these events enhance membraneand protein stability leading to increased seed longevity (Brenac et al.,1997; Obendorf, 1997; Obendorf et al., 1998). In fact, it has been proposedthat seeds having RFOs smaller than 1.0 tend to possess shorter seed storagelives while those with RFOs greater than 1.0 have longer seed storage lives(Horbowicz and Obendorf, 1994).

Despite these studies, increasing evidence suggests that RFOs may notbe involved in increasing orthodox seed storage longevity. For example, nounique relationship was found between tomato seed longevity and sucroseor oligosaccharide content (Gurusinghe and Bradford, 2001). Studies ofArabidopsis seed longevity using recombinant inbred lines differing in

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RFO content also failed to find a relationship between RFO content anddesiccation tolerance to controlled deterioration (Groot et al., 2000). Intra-cellular glass stability of impatiens and pepper seeds using electron spinprobes showed no change before and after priming despite a reduction inRFO content (Buitink, Hemminga, and Hoekstra, 2000). Since priming re-duces storage life (Argerich and Bradford, 1989), these data suggest that thestability of intracellular glasses may not be involved in increasing orthodoxseed longevity.

REPAIR OF SEED DAMAGE

Considerable evidence indicates that repair of DNA (Rao, Roberts, andEllis, 1987; Sivritepe and Dourado, 1994; Dell’Aquila and Tritto, 1990),RNA (Kalpana and Madhava Rao, 1997), protein (Dell’Aquila and Tritto,1991; Petruzzeli, 1986), membranes (Petruzzeli, 1986; Tilden and West,1985; Powell and Harman, 1985), and enzymes (Jeng and Sung, 1994) oc-curs during imbibition. Increasing seed moisture content hastens the repairprocess (Ward and Powell, 1983). Oxygen also increases the repair of high-moisture (27 to 44 percent) lettuce (Ibrahim, Roberts, and Murdoch, 1983)and high-moisture (24 to 31 percent) wheat (Petruzzeli, 1986) seeds, sug-gesting that respiratory activity is an essential component of repair. Thisknowledge that repair occurs during imbibition has been practically adaptedby the seed industry for many crops through seed priming. As a result, stud-ies examining the physiological advantages/disadvantages in extendingseed performance are appropriate. In general, it is accepted that repair ofseeds deteriorated by lipid peroxidation occurs during hydration (priming).The repaired seed is then dried for normal handling and the benefits of re-pair retained as the primed seed completes germination. It should be noted,however, that the physiological improvements gained by priming are notsolely attributable to repair since newly harvested muskmelon seeds show adramatic improvement in seed performance following osmopriming (Wel-baum and Bradford, 1991).

Most studies conclude that “repair” has occurred, but when (duringpriming or after), where (what seed part is repaired, if any), and how (whatis the mechanism) repair occurs is still not known.

When Does Repair Occur?

The time when the beneficial effects of priming are achieved is un-known. It is generally thought that the hydration phase causes activation ofessential metabolism associated with germination and the production of re-

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pair enzymes. These remain potentially active following subsequent dryingand are quickly reactivated on imbibition, culminating in more rapid anduniform completion of germination. Other studies, however, suggest thatthe maximum beneficial effects of priming are achieved during the dryingphase when enzymes are afforded sufficient time to effect repair and physi-ologically stabilize the seed. For example, the optimum effects of wheatseed osmopriming are observed two weeks after drying (Dell’Aquila andTritto, 1990). Dell’Aquila and Bewley (1989) showed that protein synthesisis reduced in the axes of pea seeds imbibed in polyetheleneglycol (PEG),dried, and then increased on their return to imbibition. Further research isnecessary to clarify whether the benefits of priming are achieved during thehydration or drying phases, or both.

Where Is the Location of Repair?

The location of the beneficial priming response still needs clarification.Reversal of seed deterioration by priming generally occurs in the meri-stematic axis or the radicle tip, e.g., peanut (Fu et al., 1988). Sivritepe andDourado (1994) found that controlled humidification of aged pea seeds to16.3 to 18.1 percent just prior to sowing decreases chromosomal aberra-tions, reduces imbibitional injury, and improves seed viability. Rao, Rob-erts, and Ellis (1987) reported a reversal of chromosomal damage (inducedduring seed aging) with partial hydration of lettuce seeds by osmoprimingto 33 to 44 percent. This treatment also increases the rate of root growth anddecreases the frequency of abnormal seedlings. In tomato, artificial agingincreases the percentage of aberrant anaphases in seedling root tips (VanPijlen et al., 1995). However, although osmopriming partially counteractsthe detrimental effects of artificial aging on germination rate, uniformity,and normal seedlings, it does not influence the frequency of aberrantanaphases in seedling root tips.

Priming also appears to increase germination metabolism in aged axesmore than those that are not aged. For example, Dell’Aquila and Taranto(1986) demonstrated that primed embryos of aged wheat seeds have a fasterresumption of cell division and DNA synthesis on subsequent imbibition.Clarke and James (1991) showed that accelerated aging has an adverse ef-fect on endosperm cells of leek seeds which results in their degradation andan overall loss in seed viability during osmopriming. During germination,however, those seeds that were aged and then osmoprimed showed an in-crease in RNA species in the whole seeds and their embryos.

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What Is the Mechanism of Repair?

Priming appears to reverse the detrimental effects of seed deterioration.In sweet corn, osmo- and matripriming results in decreased conductivity,free sugars, and DNA content, while RNA content increased (Sung andChang, 1993). Natural aging of French bean seeds stored for up to fouryears induced membrane disruption and leakage of UV-absorbing sub-stances, which was ameliorated by hydropriming (Pandey, 1988, 1989).Lower electrical conductivity readings following hydropriming indicatedreduced membrane leakage for eggplant and radish (Rudrapal and Naka-mura, 1988a) and onion (Choudhuri and Basu, 1988) seeds. These benefi-cial effects may be due to the flushing of solutes from the seed during thepriming procedure and prior to determination of leaked substances. As apractical result, primed seeds often perform better in disease-infested soilsbecause of decreased electrolyte leakage and faster germination which re-duce the window of opportunity for fungal attack (Osburn and Schroth,1988). Osmopriming increased respiration in tabasco and jalapeno seeds(Sundstrom and Edwards, 1989; Halpin-Ingham and Sundstrom, 1992), al-though respiratory rates in –1.35 MPa NaCl osmoprimed pepper seeds werethe same as in raw seeds (Smith and Cobb, 1992).

Priming is also thought to increase enzyme activity and counteract theeffects of lipid peroxidation. Saha, Mandal, and Basu (1990) showed thatmatripriming caused increased amylase and dehydrogenase activity in agedsoybean seeds compared to raw seeds. In wheat, osmopriming increasedprotein and DNA synthesis (Dell’Aquila and Tritto, 1990). L-isoaspartylmethyltransferase enzymes were reported to initiate the conversion of detri-mental L-isoaspartyl residues to normal L-isoaspartyl residues that accumu-late in naturally aged wheat seeds (Mudgett and Clarke, 1993; Mudgett,Lowenson, and Clarke, 1997). This enzyme is present in seeds of 45 speciesfrom 23 families representing most of the divisions of the plant kingdom(Mudgett, Lowenson, and Clarke, 1997). Osmoprimed tomato seeds sub-jected to accelerated aging showed restored activity of L-isoaspartyl methyl-transferase to levels similar to nonaged controls, leading Kester, Geneve,and Houtz (1997) to suggest that this enzyme is involved in early repair ofdeteriorated seeds. Osmopriming reverses the loss of lipid-peroxidation-detoxifying enzymes, such as superoxide dismutase, catalase, and gluta-thione reductase, in aged sunflower seeds, and these enzymes are present atthe same activities as in unaged seeds (Bailly et al., 1997).

Priming also reduces lipid peroxidation during subsequent seed storage.In onion seed, Choudhuri and Basu (1988) demonstrated that hydroprimingtreatments effectively slowed physiological deterioration under natural (15months) and accelerated aging conditions, with the effect being dependenton seed vigor. This improved storability was associated with greater dehy-

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drogenase activity and appreciably lower peroxide formation in cells. Simi-lar findings were reported for hydroprimed eggplant and radish seeds withthe conclusion that hydropriming reduces free radical damage to cellularcomponents (Rudrapal and Nakamura, 1988). Jeng and Sung (1994) foundthat free radical scavenging enzymes such as superoxide dismutase, cata-lase, and peroxidase and glyoxysome enzymes such as isocitrate lyase andmalate synthase were increased by increasing hydration of artificially agedpeanut seeds. Chang and Sung (1998) also showed that martripriming withvermiculite of sweet corn seeds enhanced the activities of several lipid per-oxide scavenging enzymes. Chiu, Wang, and Sung (1995) found that in-creasing hydration enhanced membrane repair in watermelon seeds andattributed this to the stimulation of peroxide scavenging enzymes that pro-duced reduced glutathione which may control aging by counteracting lipidperoxidation. Another possible antioxidant is glutathione whose contenthas been shown to decrease with watermelon seed aging as seeds are hy-drated (Hsu and Sung, 1997).

MODEL OF SEED DETERIORATION AND REPAIRDURING PRIMING/HYDRATION

As seeds deteriorate, a cascade of disorganization ensues, ultimately lead-ing to complete loss of cell function. The current model of seed deteriorationaccepts lipid peroxidation as a central cause of cellular degeneration throughfree radical assault on important cellular molecules and structures. Figure 9.5demonstrates some proposed events associated with seed deterioration dur-ing storage and their repair, or lack of repair, during hydration that can occurduring imbibition or priming and seeds contain a variety of antioxidants in-cluding vitamins, polyphenols, and flavonoids (Larson, 1997).

Storage

Low seed moisture content during storage favors free radical productionby autoxidation. Through lipid peroxidation, these free radicals either di-rectly or indirectly cause four types of cellular damage: mitochondrial dys-function, enzyme inactivation, membrane perturbations, and genetic dam-age. Thus, the amount of antioxidants in seeds might reduce the incidenceof cellular damage due to free radical assault during seed storage.

Imbibition and Priming

As time of seed storage increases, so does cellular damage. Imbibitionand priming of the seed allows two events to occur. As imbibition proceeds,

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the cascade of cellular damage caused by autoxidation is furthered by freeradical damage, induced less by autoxidation and more by free-radical-generating hydrolytic enzymes such as lipoxygenase. The presence of anti-oxidants may ameliorate this damage. In addition, upon hydration, anabolicenzymes associated with repair of cellular constituents counter these de-generative events. Their success determines whether a seed is capable ofgerminating and performing optimally. If unsuccessful, the cellular damageestablished during storage leads to unalterable detrimental physiologicalconsequences resulting in a nongerminable seed.

CONCLUSIONS

In conclusion, this chapter has emphasized that many factors (externaland internal) contribute to orthodox seed deterioration. Of these, seed mois-ture content and temperature have important roles that directly influencethe biochemistry of deterioration. It is also apparent that seed deteriorationis uniform neither among seeds nor among seed parts (membranes being

GerminableSeed

NongerminableSeed

FIGURE 9.5. A model of seed deterioration and its physiological consequencesduring seed storage and imbibition (Source: M. B. McDonald, 1999, Seed deteri-oration: Physiology, repair and assessment, Seed Science and Technology 27:177-237. Reproduced with permission.)

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more prone to deteriorative events). At the cellular level, the mitochondriamay be a central organelle susceptible to deteriorative event, and their fur-ther study is warranted. As oxygen “sinks” that contain extensive mem-brane structure for respiratory events, they are particularly prone to free rad-ical assault and lipid peroxidation. If these events occur, seed germinationas measured by speed and uniformity of emergence would certainly becompromised. Fortunately, evidence exists that free radical attack can be re-duced by free radical scavenger and antioxidant compounds found in seeds.In addition, specific repair enzymes have been identified that potentiallyfunction during hydration, perhaps providing a mechanism for the successof priming as a seed-enhancement technology. Clearly, all of this demon-strates that further studies are necessary to better understand the mecha-nism(s) of orthodox seed deterioration and its repair. Hopefully, this chapterhas provided the foundation to initiate this quest.

REFERENCES

Argerich, C.A. and Bradford, K.J. (1989). The effects of priming and aging on seedvigor in tomato. Journal of Experimental Botany 40: 599-607.

Association of Official Seed Analysts (AOSA) (2000). Tetrazolium Testing Hand-book. Contribution No. 29. Lincoln, NE: Author.

Bailly, C., Benamar, A., Corbineau, F., and Côme, D. (1997). Changes in super-oxide dismutase, catalase and glutathione reductase activities in sunflower seedsduring accelerated aging and subsequent priming. In Ellis, R.H., Black, M.,Murdoch, A.J., and Hong, T.D. (Eds.), Basic and Applied Aspects of Seed Biol-ogy (pp. 665-672). Boston: Kluwer Academic Publishers.

Barnes, W.M. (1994). PCR amplification of up to 35-kb DNA with high fidelity andhigh yield from lambda bacteriophage templates. Proceedings of the NationalAcademy of Sciences, USA 91: 2216-2220.

Beckman, K.B. and Ames, B.N. (1998). The free radical theory of aging matures.Physiological Reviews 78: 547-581.

Berjak, P., Dini, M., and Gevers, H.O. (1986). Deteriorative changes in embryos oflong-stored, uninfected maize caryopses. South African Journal of Botany 52:109-116.

Berjak, P., Farrant, J.M., Mycock, D.J., and Pammenter, N.W. (1990). Recalcitrant(homoiohydrous) seeds: The enigma of their desiccation sensitivity. Seed Sci-ence and Technology 18: 297-310.

Bewley, J.D. (1986). Membrane changes in seeds as related to germination and theperturbations resulting from deterioration in storage. In McDonald, M.B. andNelson, C.J. (Eds.), Physiology of Seed Deterioration (pp. 27-47). Madison, WI:Crop Science Society of America.

Bhattacharjee, A. and Bhattacharyya, R.N. (1989). Prolongation of seed viability ofOryza sativa L. cultivar Ratna by dikegulac-sodium. Seed Science and Technol-ogy 17: 309-316.

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Bhattacharjee, A., Chowdhury, R.S., and Choudhuri, M.A. (1986). Effects of CCCand Na-dikegulac on longevity and viability of seeds of two jute cultivars. SeedScience and Technology 14: 127-139.

Bhattacharjee, A. and Gupta, K. (1985). Effect of dikegulac-sodium, and growth re-tardant, on the viability of sunflower seeds. Seed Science and Technology 13:165-174.

Bingham, I.J., Harris, A., and MacDonald, L. (1994). A comparative study of radi-cle and coleoptile extension in maize seedlings from aged and unaged seeds.Seed Science and Technology 22: 127-139.

Brenac, P., Horbowics, M., Downer, S.M., Dickerman, A.M., Smith, M.E., andObendorf, R.L. (1997). Raffinose accumulation related to desiccation toleranceduring maize (Zea mays L.) seed development and maturation. Journal of PlantPhysiology 15: 481-488.

Buitink, J., Hemminga, M.A., and Hoekstra, F.A. (2000). Is there a role for oligo-saccharides in seed longevity? An assessment of intracellular glass stability.Plant Physiology 122: 1217-1224.

Buitink, J., Leprince, O., Hemminga, M.A., and Hoekstra, F.A. (2000). Molecularmobility in the cytoplasm: An approach to describe and predict lifespan of drygermplasm. Proceedings of the National Academy of Sciences, USA 97: 2385-2390.

Burke, M.J. (1986). The glassy state and survival of anhydrous biological systems.In Leopold, A.C. (Ed.), Membranes, Metabolism and Dry Organisms (pp. 358-363). Ithaca, NY: Cornell University Press.

Chang, S.M. and Sung, J.M. (1998). Deteriorative changes in primed sweet cornseeds during storage. Seed Science and Technology 26: 613-626.

Chauhan, K.P.S. (1985). The incidence of deterioration and its localization in agedseeds of soybean and barley. Seed Science and Technology 13: 769-773.

Cheng, S., Higuchi, R., and Stoneking, M. (1994). Complete mitochondrial genomeamplification. Nature Genetics 7: 350-351.

Chhetri, D.R., Rai, A.S., and Bhattacharjee, A. (1993). Chemical manipulation ofseed longevity of four crop species in an unfavorable storage environment. SeedScience and Technology 21: 31-44.

Chin, H.F. and Roberts, E.H. (1980). Recalcitrant Crop Seeds. Kuala Lumpur, Ma-laysia: Tropical Press.

Chiu, K.Y., Wang, C.S., and Sung, J.M. (1995). Lipid peroxidation and peroxide-scavenging enzymes associated with accelerated aging and hydration of water-melon seeds differing in ploidy. Physiologia Plantarum 94: 441-446.

Choudhuri, N. and Basu, R.N. (1988). Maintenance of seed vigour and viability ofonion (Allium cepa L.). Seed Science and Technology 16: 51-61.

Chowdhury, S.R. and Choudhuri, M.A. (1994). Effects of seed pretreatment withCCC, cinnamic acid, and Na-dikegulac on germination and early seedlinggrowth performance from ageing jute seeds under water deficit stress. Seed Sci-ence and Technology 22: 203-208.

Clarke, N.A. and James, P.E. (1991). The effects of priming and acclerated ageingupon the nucleic acid content of leek seeds and their embryos. Journal of Experi-mental Botany 42: 261-268.

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Conley, C.A. and Hanson, M.R. (1995). How do alterations in plant mitochondrialgenomes disrupt pollen development? Journal of Bioenergetics and Biomem-branes 27: 447-457.

Copeland, L.O. and McDonald, M.B. (2001). Principles of Seed Science and Tech-nology. New York: Kluwer Academic Press.

Crowe, J.H., Crowe, L.M., and Hoekstra, F.A. (1989). Phase transitions and perme-ability changes in dry membranes during rehydration. Journal of Bioenergeticsand Biomembranes 21: 77-91.

Crowe, J.H., Hoekstra, F.A., and Crowe, L.M. (1992). Anhydrobiosis. Annual Re-views of Physiology 54: 579-599.

Das, G. and Sen-Mandi, S. (1988). Root formation in deteriorated (aged) wheat em-bryos. Plant Physiology 88: 983-986.

Das, G. and Sen-Mandi, S. (1992). Triphenyl tetrazolium chloride staining pattern ofdifferentially aged wheat embryos. Seed Science and Technology 20: 367-373.

DeGrey, A.D.W.J. (1999). The Mitochondrial Free Radical Theory of Aging. Aus-tin, TX: R.G. Landes Company.

Dell’Aquila, A. and Bewley, J.D. (1989). Protein synthesis in the axes of polyethyl-ene glycol treated pea seed and during subsequent germination. Journal of Ex-perimental Biology 40: 1001-1007.

Dell’Aquila, A. and Taranto, G. (1986). Cell division and DNA synthesis duringosmoconditioning treatment and following germination in aged wheat embryos.Seed Science and Technology 14: 333-341.

Dell’Aquila, A. and Tritto, V. (1990). Ageing and osmotic priming in wheat seeds:Effects upon certain components of seed quality. Annals of Botany 65: 21-26.

Dell’Aquila, A. and Tritto, V. (1991). Germination and biochemical activities inwheat seeds following delayed harvesting, ageing and osmotic priming. SeedScience and Technology 19: 73-82.

DePaula, M., Perez-Otaola, M., Darder, M., Torres, M., Frutos, G., and Martinez-Honduvilla, C.J. (1996). Function of the ascorbate-glutathione cycle in agedsunflower seeds. Physiologia Plantarum 96: 543-550.

DeVos, C.H.R., Kraak, H.L., and Bino, R.J. (1994). Ageing of tomato seeds in-volves glutathione oxidations. Physiologia Plantarum 92: 131-139.

Dey, P.G. and Mukherjee, R.K. (1988). Invigoration of dry seeds with physiologi-cally active chemicals in organic solvents. Seed Science and Technology 16:145-153.

Ellis, R.H., Osei-Bonsu, K., and Roberts, E.H. (1982). The influence of genotype,temperature, and moisture on seed longevity in chickpea, cowpea, and soyabean. Annals of Botany 50: 69-82.

Esashi, Y., Kamataki, A., and Zhang, M. (1997). The molecular mechanism of seeddeterioration in relation to the accumulation of protein-acetaldehyde adducts. InEllis, R.H., Black, M., Murdoch, A.J., and Hong, T.D. (Eds.), Basic and AppliedAspects of Seed Biology (pp. 489-498). Boston: Kluwer Academic Publishers.

Fu, J.R., Lu, X.H., Chen, R.Z., Zhang, B.Z., Liu, Z.S., Ki, Z.S., and Cai, C.Y.(1988). Osmoconditioning of peanut (Arachis hypogaea L.) seeds with PEG toimprove vigour and some biochemical activities. Seed Science and Technology16: 197-212.

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Garwood, N.C. (1989). Tropical soil seed banks: A review. In Leck, M.A., Parker,V.T., and Simpson, R.L. (Eds.), Ecology of Soil Seed Banks (pp. 149-209). SanDiego, CA: Academic Press.

Gille, J.J.P. and Joenje, H. (1991). Biological significance of oxygen toxicity: Anintroduction. In Vigo-Pelfrey, C. (Ed.), Membrane Lipid Oxidation (pp. 1-32).Boca Raton, FL: CRC Press.

Groot, S.P.C., van der Geest, A.H.M., Tesnier, K., Alonso-Blanco, C., Bentsink, L.,Donkers, H., Koornneef, M., Vreugdenhil, D., and Bino, R.J. (2000). Moleculargenetic analysis of Arabidopsis seed quality. In Black, M., Bradford, K.J., andVazquez-Ramos, J. (Eds.), Seed Biology: Advances and Implications (pp. 123-132). Wallingford, UK: CABI Publishing.

Gurusinghe, S. and Bradford, K.J. (2001). Galactosyl-sucrose oligosaccharides andpotential longevity of primed seeds. Seed Science Research 11: 121-133.

Hahalis, D.A. and Smith, M.L. (1997). Comparison of the storage potential ofsoyabean (Glycine max) cultivars with different rates of water uptake. In Ellis,R.H., Black, M., Murdoch, A.J., and Hong, T.D. (Eds.), Basic and Applied As-pects of Seed Biology (pp. 507-514). Boston: Kluwer Academic Publishers.

Hailstones, M.D. and Smith, M.T. (1991). Soybean seed invogoration by ferroussulfate: Changes in lipid peroxidation, conductivity, tetrazolium reduction, DNAand protein synthesis. Journal of Plant Physiology 137: 307-311.

Halmer, P. and Bewley, J.D. (1984). A physiological perspective on seed vigourtesting. Seed Science and Technology 12: 561-575.

Halpin-Ingham, B. and Sundstrom, F.J. (1992). Pepper seed water content, germi-nation response and respiration following priming treatments. Seed Science andTechnology 20: 589-596.

Hanson, M.R. and Otto, F. (1992). Structure and function of the higher plant mito-chondrial genome. International Review of Cytology 141: 129-172.

Harrington, J.F. (1972). Seed storage and longevity. In Kozlowski, T.T. (Ed.), SeedBiology, Volume 3 (pp. 145-240). New York: Academic Press.

Hoekstra, F.A., Crowe, J.H., and Crowe, L.M. (1992). Germination and ion leakageare linked with phase transitions of membrane lipids during imbibition of Typhalatifolia pollen. Physiologia Plantarum 84: 29-34.

Horbowicz, M. and Obendorf, R.L. (1994). Seed desiccation tolerance and stor-ability: Dependence on flatulence-producing oligosaccharides and cyclitols—Review and survey. Seed Science Research 4: 385-405.

Hsu, J.L. and Sung, J.M. (1997). Antioxidant role of glutathione associated with ac-celerated aging and hydration of triploid watermelon seeds. Physiologia Plant-arum 100: 967-974.

Ibrahim, A.E., Roberts, E.H., and Murdoch, A.J. (1983). Viability of lettuce seeds:II. Survival and oxygen uptake in somatically controlled storage. Journal of Ex-perimental Botany 34: 631-640.

Jeng, T.L. and Sung, J.M. (1994). Hydration effect on lipid peroxidation and perox-ide-scavenging enzymes activity of artificially-aged peanut seed. Seed Scienceand Technology 22: 531-539.

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Kadenbach, B., Munscher, C., and Frank, V. (1995). Human aging is associatedwith stochastic somatic mutations of mitochondrial DNA. Mutation Research338: 161-172.

Kalpana, R. and Madhava Rao, K.V. (1994). Absence of the role of lipid perox-idation during accelerated aging of seeds of pigeonpea [Cajanus cajan (L.)Millsp.] cultivars. Seed Science and Technology 22: 253-260.

Kalpana, R. and Madhava Rao, K.V. (1997). Nucleic acid metabolism of seeds ofpegeonpea (Cajanus cajan L. Millsp.) cultivars during accelerated ageing. SeedScience and Technology 25: 293-301.

Kang, D., Takeshige, K., Sekiguchi, M., and Singh, K.K. (1998). Introduction. InSingh, K.K. (Ed.), Mitochondrial DNA Mutations in Aging, Disease and Cancer(pp. 1-15). New York: Springer.

Kester, S.T., Geneve, R.L., and Houtz, R.L. (1997). Priming and accelerated agingaffect L-isoaspartyl methyltransferase activity in tomato (Lycopersicon escul-entum Mill) seed. Journal of Experimental Botany 48: 943-949.

Larson, R.A. (1997). Naturally Occurring Antioxidants. Boca Raton, FL: LewisPublishers.

Loiseau, J., Benoit, L.V., Macherel, M.H., and Deunff, Y.L. (2001). Seed lipoxy-genases: Occurrence and functions. Seed Science Research 11: 199-211.

Lu, B., Wilson, R.K., Phreaner, C.G., Mulligan, M.R., and Hanson, M.R. (1996).Protein polymorphism generated by differential RNA editing of a plant mito-chondrial rps12 gene. Molecular and Cellular Biology 16: 1543-1549.

McDonald, M.B. (1985). Physical seed quality of soybean. Seed Science and Tech-nology 13: 601-628.

McDonald, M.B. (1999). Seed deterioration: Physiology, repair and assessment.Seed Science and Technology 27: 177-237.

McDonald, M.B. and Nelson, C.J. (1986). Physiology of Seed Deterioration. CSSASpecial Publication No. 11. Madison, WI: Crop Science Society of America.

McDonald, M.B., Sullivan, J., and Lauer, M.J. (1994). The pathway of water uptakein maize (Zea mays L.) seeds. Seed Science and Technology 22: 79-90.

McDonald, M.B., Vertucci, C.W., and Roos, E.E. (1988). Soybean seed imbibition:Water absorption of seed parts. Crop Science 28: 993-998.

Mudgett, M.B. and Clarke, S. (1993). Characterization of plant L-isoaspartyl meth-yltransferases that may be involved in seed survival: Purification, cloning, andsequence analysis of the wheat germ enzyme. Biochemistry 32: 111000-111111.

Mudgett, M.B., Lowenson, J.D., and Clarke, S. (1997). Protein repair L-isoaspartylmethyltransferase in plants: Phylogenetic distribution and the accumulation ofsubstrate proteins in aged barley seeds. Plant Physiology 114: 1481-1489.

Munscher, C., Muller-Hocker, J., and Kadenbach, B. (1993). Human aging is asso-ciated with various point mutations in tRNA genes of mitochondrial DNA. Bio-logical Chemistry Hoppe Seyler 374: 1099-1104.

Newton, K.J. and Gabay-Laughman, S.J. (1998). Abnormal growth and male steril-ity associated with mitochondrial DNA arrangements in plants. In Singh, K.K.(Ed.), Mitochondrial DNA Mutations in Aging, Disease, and Cancer (pp. 365-381). New York: Springer.

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Obendorf, R.L. (1997). Oligosaccharides and galactosyl cyclitols in seed desicca-tion tolerance. Seed Science Research 7: 63-74.

Obendorf, R.L., Dickerman, A.M., Pflum, T.M., Kacalanos, M.A., and Smith, M.E.(1998). Drying rate alters soluble carbohydrates, desiccation tolerance, and sub-sequent seedling growth of soybean (Glycine max L. Merrill) zygotic embryos invitro maturation. Plant Science 132: 1-12.

Oliver, A.E., Crowe, L.M., and Crowe, J.H. (1998). Methods for dehydration-toler-ance: Depression of the phase transition temperature in dry membranes and car-bohydrate vitrification. Seed Science Research 8: 211-221.

Osburn, R.M. and Schroth, M.N. (1988). Effect of osmopriming sugar beet seed onexudation and subsequent damping-off caused by Pythium ultimum. Phyto-pathologie 78: 1246-1250.

Pammenter, N.W. and Berjak, P. (2000). Evolutionary and ecological aspects of re-calcitrant seed biology. Seed Science Research 10: 301-306.

Pandey, K.K. (1988). Priming induced repair in French bean seeds. Seed Scienceand Technology 16: 527-532.

Pandey, K.K. (1989). Priming induced alleviation of the effects of natural ageingderived selective leakage of constituents in French bean seeds. Seed Science andTechnology 17: 391-397.

Peterbauer, T. and Richter, A. (2001). Biochemistry and physiology of raffinosefamily oligosaccharides and galactosyl cyclitols in seeds. Seed Science Research11: 185-197.

Petruzzeli, L. (1986). Wheat viability at high moisture content under hermetic andaerobic storage conditions. Annals of Botany 58: 259-265.

Powell, A.A. and Harman, G.E. (1985). Absence of a consistent association ofchanges in membranal lipids with the ageing of pea seeds. Seed Science andTechnology 13: 659-667.

Priestley, D.A. (1986). Seed Ageing: Implications for Seed Storage and Persistencein the Soil. Ithaca, NY: Cornell University Press.

Rao, N.K., Roberts, E.H., and Ellis, R.H. (1987). Loss of viability in lettuce seedsand the accumulation of chromosome damage under different storage condi-tions. Annals of Botany 60: 85-96.

Rasmussen, L.J. and Singh, K.K. (1998). Genetic integrity of the mitochondrial ge-nome. In Sing, K.K. (Ed.), Mitochondrial DNA Mutations in Aging, Disease,and Cancer (pp. 115-127). New York: Springer.

Reynier, P. and Mathiery, Y. (1995). Accumulation of deletions in mtDNA duringtissue aging: Analysis by long PCR. Biochemical and Biophysical ResearchCommunications 217: 59-67.

Rudrapal, D. and Nakamura, S. (1988a). The effect of hydration-dehydrationpretreatments on eggplant and radish seed viability and vigour. Seed Science andTechnology 16: 123-130.

Rudrapal, D. and Nakamura, S. (1988b). Use of halogens in controlling eggplantand radish seed deterioration. Seed Science and Technology 16: 115-122.

Saha, R., Mandal, A.K., and Basu, R.N. (1990). Physiology of seed invigorationtreatments in soybean (Glycine max L.). Seed Science and Technology 18: 269-276.

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Sembdner, G. and Parthier, B. (1993). The biochemistry and the physiological andmolecular actions of jasmonates. Annual Review of Plant Physiology and PlantMolecular Biology 44: 569-589.

Seneratna, T., Gusse, J.F., and McKersie, B.D. (1988). Age-induced changes in cel-lular membranes of imbibed soybean axes. Physiologia Plantarum 73: 85-91.

Sivritepe, H.O. and Dourado, A.M. (1994). The effects of humidification treatmentson viability and the accumulation of chromosomal aberrations in pea seeds. SeedScience and Technology 22: 337-348.

Smith, M.T. and Berjak, P. (1995). Deteriorative changes associated with the loss ofviability of stored desiccation-tolerant and desiccation-sensitive seeds. In Kigel,J. and Galili, G. (Eds.), Seed Development and Germination (pp. 701-746). NewYork: Marcel Dekker.

Smith, P.T. and Cobb, B.G. (1992). Physiological/enzymatic characteristics ofprimed, redried, and germinated pepper seeds (Capsicum annuum L.). Seed Sci-ence and Technology 20: 503-513.

Society of Commercial Seed Technologists (SCST) (2001). Seed TechnologistsTraining Manual. Lincoln, NE: Author.

Staswick, P.E. (1992). Jasmonate, genes and fragrant signals. Plant Physiology 99:804-807.

Sundstrom, F.J. and Edwards, R.L. (1989). Pepper seed respiration, germination,and seedling development following seed priming. HortScience 24: 343-345.

Sung, F.J.M. and Chang, Y.H. (1993). Biochemical activities associated with prim-ing of sweet corn seeds to improve vigor. Seed Science and Technology 21: 97-105.

Suzuki, Y., Ise, K., Li, C.Y., Honda, I., Iwai, Y., and Matsukura, U. (1999). Volatilecomponents in stored rice [Oryza sativa (L.)] of volatiles with and withoutlipoxygenase-3 in seeds. Journal of Agricultural and Food Chemistry 47: 1119-1124.

Suzuki, Y., Yasui, T., Matsukura, U., and Terao, J. (1996). Oxidative stability ofbran lipids from rice variety [Oryza sativa (L.)] lacking lipoxygenase-3 in seeds.Journal of Agricultural and Food Chemistry 44: 3479-3483.

Tarquis, A.M. and Bradford, K.J. (1992). Prehydration and priming treatments thatadvance germination also increase the rate of deterioration of lettuce seeds. Jour-nal of Experimental Botany 43: 307-317.

Tilden, R.L. and West, S.H. (1985). Reversal of the effects of ageing in soybeanseeds. Plant Physiology 77: 584-586.

Van Pijlen, J.G., Kraak, H.L., Bino, R.J., and De Vos, C.H.R. (1995). Effects of age-ing and osmoconditioning on germination characteristics and chromosome aber-rations of tomato (Lycopersicon esculentum Mill.) seeds. Seed Science andTechnology 23: 823-830.

Vazquez-Yanes, C. and Orozco-Segovia, A. (1993). Patterns of seed longevity andgermination in the tropical rainforest. Annual Review of Ecology and Systemat-ics 24: 69-87.

Ward, F.H. and Powell, A.A. (1983). Evidence for repair processes in onion seedsduring storage at high seed moisture contents. Journal of Experimental Botany34: 277-282.

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Welbaum, G.E. and Bradford, K.J. (1991). Water relations of seed development andgermination in muskmelon (Cucumis melo L.): VI. Influence of priming on ger-mination responses to temperature and water potential during seed development.Journal of Experimental Botany 42: 393-399.

Wilson, D.O. and McDonald, M.B. (1986a). A convenient volatile aldehyde assayfor measuring seed vigour. Seed Science and Technology 14: 259-268.

Wilson, D.O. and McDonald, M.B. (1986b). The lipid peroxidation model of seeddeterioration. Seed Science and Technology 14: 269-300.

Wolkers, W.F., Bochicchio, A., Selvaggi, G., and Hoekstra, F.A. (1998). Fouriertransform infrared microspectroscopy detects changes in protein secondarystructure associated with desiccation tolerance in developing maize embryos.Plant Physiology 116: 1169-1177.

Zacheo, G., Cappello, A.R., Perrone, L.M., and Gnoni, G.V. (1998). Analysis offactors influencing lipid oxidation of almond seeds during accelerated aging.Lebensmittel-Wissenschaft und Technologie 31: 6-9.

Zimmerman, D.C. and Coudron, C.A (1979). Identification of traumatin, a woundhormone, as 12-oxo-trans-10-dodecenoic acid. Plant Physiology 63: 536-541.

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Chapter 10

Recalcitrant SeedsRecalcitrant Seeds

Patricia BerjakNorman W. Pammenter

SEED CHARACTERISTICS—THE BROAD PICTURE

Aside from the provision of food and feedstock from one season to thenext, seeds are stored as base and active collections, as a means of long-termconservation of valuable genetic resources representing species biodiversity,and to provide planting stock for subsequent seasons. However, their con-servation in seed banks or gene banks, or in commercial storage, makes theassumption that seeds are storable in the first place, which in turn is basedon the premise that they show orthodox postharvest behavior (Roberts,1973). By this it is meant that the period for which the seeds may be storedwithout loss of quality is predictable under defined conditions of storagetemperature and seed water (moisture) content, the longevity, within limits,increasing logarithmically with decreasing water content (Ellis and Rob-erts, 1980). Orthodox seeds are, or can be, dehydrated to low water con-tents, which is a consequence of their having acquired the property of desic-cation tolerance relatively early during their preshedding development(e.g., Bewley and Black, 1994; Vertucci and Farrant, 1995). The property ofdesiccation tolerance and its maintenance in dry orthodox seeds is based onthe presence and interplay of a suite of mechanisms and processes ex-pressed during development (Pammenter and Berjak, 1999).

However, not all seeds are orthodox—i.e., some seeds do not fully ac-quire the property of desiccation tolerance. Indeed, the responses to dehy-dration of mature seeds of some species (e.g., Avicennia marina, Farrant,Berjak, and Pammenter, 1993; Farrant, Pammenter, and Berjak, 1993) indi-cate that few, if any, of the mechanisms and processes allowing tolerance ofthe loss of more than the slightest proportion of tissue water are operational.Such seeds are highly recalcitrant—this being the term introduced by Rob-erts (1973) for seeds that cannot be stored at low water contents. Since thepublication by Chin and Roberts (1980), the list of species recorded as pro-

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ducing recalcitrant seeds—or, at any rate, seeds that are nonorthodox(orthodox seeds being those that are desiccation tolerant)—has steadilylengthened. Some examples of species producing recalcitrant seeds thathave been recorded over the past decade alone include Machilus thunbergiiand M. kusanoi (Lin and Chen, 1995 and Chien and Lin, 1997, respec-tively); Aporusa lindleyana (Kumar, Thomas, and Pushpangadan, 1996);Garcinia gummi-gutta (Chacko and Pillai, 1997); Litsea acuminata (Chienand Yang, 1997); Euterpe edulis (de Andrade and Pereira, 1997); possiblysome species of dryland palms from Africa and Madagascar (Davies andPritchard, 1998); Carapa guianensis and C. procera (Connor et al., 1998);Guarea guidonia (Connor and Bonner, 1998); Inga uruguensis (Bilia,Marcos Filho, and Novembre, 1999); Bhesa indica (Kumar and Chacko,1999); and Boscia senegalensis (an arid-zone species), Butyrospermumparkii, Cordyla pinnata, and Saba senegalensis (Danthu et al., 2000).

Several points arise from this list: first, the genera and species concernedwill be largely unfamiliar; second, although not stated, their provenance istropical or subtropical; and third—also not immediately obvious—is that,as far as can be ascertained, all are tree species. These points, in turn, under-lie three generalizations: (1) almost all the knowledge amassed to dateabout seed biology and physiology has been derived from work on culti-vated crops plus a few woody species, representing less than 0.1 percent ofthe higher plants, which is hardly a representative sample of the more than250,000 documented species of spermatophytes; (2) probably many tropi-cal and subtropical species produce nonorthodox seeds; and (3) such seedsappear to be produced predominantly by trees (Berjak and Pammenter,2001), although nonorthodox seeds produced by herbaceous species havebeen recorded, especially among the Amaryllidaceae.

SEED BEHAVIOR

Evolutionary and Taxonomic Considerations

There seems to be little correlation between the occurrence of seed recal-citrance—considered simply as desiccation sensitivity—and taxonomicstatus, as the phenomenon is widespread across families. Although thereare dicotyledonous families in which apparently no species produces recal-citrant seeds, in others the phenomenon is common (e.g., the Dipterocar-paceae, Tompsett, 1992). In an in-depth review, von Teichman and van Wyk(1994) associated recalcitrance across 45 dicotyledonous families withlarge seeds developing from bitegmic, crassinucellate ovules showing nu-clear endosperm development. Those authors also drew attention to the

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woody habitat and tropical habitat being associated features which, to-gether with the ovule/seed characteristics, are generally considered as an-cestral states. However, recalcitrant seeds are also produced by species inrelatively advanced dicotyledonous families, as well as by some that aremonocotyledonous. Despite the opinion that characteristics of contempo-rary seeds probably reflect an evolutionary history incorporating parallel-ism, convergence, and reversion, when the evidence is weighed up recalci-trance is considered to be the ancestral seed condition in the angiosperms(von Teichman and van Wyk, 1994; Pammenter and Berjak, 2000).

It seems probable that production of recalcitrant seeds (whether as anancestral or relictual trait or by reversion) has been favored in environmentswhere there would be little selective advantage to the acquisition of desicca-tion tolerance, e.g., in the humid tropics or other regions where no seasonalconstraints prevent immediate seedling establishment. Nevertheless, recal-citrant seeds are produced by some species in dry environments, e.g.,Boscia senegalensis from the Sahelian zone (Danthu et al., 2000), Vitellariaparadoxum (Butyrospermum paradoxum) from Burkina Faso (Gamene,1997), and possibly some dryland palms (Davies and Pritchard, 1998), al-though surprisingly, of 87 aquatic species only 6.9 percent were unequivo-cally established as producing seeds showing recalcitrant storage behavior(Hay et al., 2000). Furthermore, a few temperate tree species also producerecalcitrant seeds which, in some cases, may overwinter in a dormant condi-tion (e.g., Aesculus hippocastanum, Pritchard, Tompsett, and Manger, 1996;Pritchard et al., 1999). These observations emphasize that a far deeper ap-preciation of seed behavior than indicated only by desiccation sensitivity isrequired and that the seed biology of many more species across the range offamilies needs to be characterized in fine detail.

Although perusal of the literature reveals that differences are apparent inthe degree of dehydration tolerated among recalcitrant seeds of differentspecies, it is difficult to make direct comparisons, as the conditions underwhich drying was carried out lack consistency. However, major differencesare also apparent in responses of seeds of different species to dehydrationunder similar conditions. A documented case involves a comparison ofthose of Araucaria angustifolia (a gymnosperm), Scadoxus membranaceus(monocotyledonous), and a dicotyledonous vine, Landolphia kirkii (Far-rant, Pammenter, and Berjak, 1989), all of which graphically illustrated le-thal intracellular responses to dehydration under identical conditions occur-ring at considerably different water contents among the species. That reportcompared seeds of completely unrelated taxa, but other record differencesin response among species of the same genus. For example, in a comparisonof intact oak seed drying responses, Connor and Bonner (1996) found thatacorns of Quercus alba were considerably more desiccation sensitive than

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were those of Q. nigra. Similarly, Normah, Ramiya, and Gintangga (1997)reported noteworthy differences in the lowest water content to which seedsof two species of Baccaurea would survive. There are also examples ofmore extreme divergence in seed behavior among species of a single genus.In the case of Acer, seeds of A. pseudoplatanus (sycamore) are sufficientlydesiccation sensitive to be unequivocally categorized as recalcitrant, whereasthose of A. platanoides (Norway maple) are orthodox (Hong and Ellis,1990). We have found a similarly wide divergence from orthodox to recalci-trant seed characteristics among southern African species of the gymno-sperm Podocarpus (our unpublished observations). For species of Coffea,Hong and Ellis (1995) describe seeds of C. liberica as showing recalcitrantpostharvest behavior, with those of C. canephora (robusta coffee) fitting thecategorization of intermediate, originally described for C. arabica (i.e., be-ing somewhat less desiccation sensitive than orthodox seeds, but showingchilling sensitivity when dehydrated, Ellis and Hong, 1990). Similarly, Eiraand colleagues (1999) described seeds of C. liberica as being the least toler-ant to dehydration with another species studied, C. racemosa, being rela-tively the most tolerant. In a study on Coffea seeds of nine species from var-ious central African provenances, Dussert and colleagues (2000) suggestedthat the varying degrees of desiccation sensitivity occurring among themcould be an adaptive feature, related to the mean number of dry months typ-ical of each habitat.

Variability

It is very difficult to define succinctly the exact nature of recalcitrance—or, more generally, of nonorthodoxy—because of the marked differences inbehavior of seeds among species, although all are shed at relatively to veryhigh water contents and all are metabolically active when shed. Inherentvariation among seeds of different species—including size, structure, thenature of the testa/pericarp, and chemical makeup—contributes to the dif-ferences in their responses to dehydration. For seeds of any one angiospermspecies, differences may also exist between the axis and cotyledons. Axeshave been shown to be more sensitive than cotyledons in Quercus robur(Finch-Savage et al., 1992) and Theobroma cacao (Li and Sun, 1999),while the reverse was found for Castanea sativa (Leprince, Buitink, andHoekstra, 1999). Our unpublished data show that for recalcitrant seeds froma spectrum of species, the axes are generally at a higher water content thanare the storage tissues, and Pritchard and colleagues (1995) reported unevendistribution of water within the component tissues of embryos of the gym-nosperm Araucaria hunsteinii. In a biophysical consideration of Coffea

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spp., Eira, Walters, and Caldas (1999) found that although the heats of sorp-tion calculated for whole seeds were similar to those of orthodox seeds, atthe same relative humidity (RH) heats of sorption for excised embryonicaxes were intermediate between values for orthodox and recalcitrant axes.However, the situation is further complicated because, for seeds of any onespecies, there is also intra- and interseasonal variation!

Intraseasonal variation will be considered later, but in terms of inter-season variation we have found that for ostensibly mature seeds of Camelliasinensis, embryonic axis water content varied from 2.0 ± 0.3 to 4.4 ± 2.4 gper g dry mass (g·g–1) for harvests made in different years (Berjak et al.,1996). Many recalcitrant seeds will show visible signs of germination instorage at the water content at which they are shed. However, seeds ofQ. robur collected in one particular year (from the same parent tree usedpreviously and since) had water contents lower than normal and did not ger-minate in storage (Finch-Savage et al., 1993; Finch-Savage, 1996). Inter-seasonal differences in germination capacity following dormancy-breakingchilling have been recorded for Aesculus hippocastanum seeds, this effectbeing ascribed to differences in mean temperature during seed filling (Prit-chard et al., 1999). Interseasonal variability is also a common feature of re-calcitrant seeds from species of tropical provenance. In two recently re-ported examples, del Carmen Rodriguez and colleagues (2000) relatedeffects of dehydration on germination with seasonality for neotropical rainforest species in Mexico, and differences in a variety of traits among seedlots of Euterpe edulis from one season to the next have also been described(Martins, Nakagawa, and Bovi, 2000).

The State of Metabolic Activity May Vary, but It Is Continuous

Familiarity with the history of any batch of recalcitrant seeds from har-vest is essential in assessing their germination performance, as well as whenattempting to explain the results of any manipulations carried out on thoseseeds (Berjak, Farrant, and Pammenter, 1989). This is because such seedsare not only hydrated, but also metabolically active. Their status is inexora-bly changing, and the state of development of recalcitrant seeds—both be-fore and after harvest—influences their desiccation sensitivity. For mostspecies of recalcitrant seeds, the least desiccation-sensitive stage occurswhen the metabolic rate is at its lowest (which generally coincides with nat-ural shedding), but they are always metabolically active (reviewed byPammenter and Berjak, 1999). If germination commences rapidly after har-vest, the seeds will show heightened sensitivity to water loss in a very shorttime. This is because as germinative metabolism progresses to the stage

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when mitosis and cellular expansion by vacuolation occur, the seeds musttake up water from an exogenous supply—and so the minimum water levelcommensurate with viability retention increases (Farrant, Pammenter, andBerjak, 1986; Berjak, Farrant, and Pammenter, 1989). Enhanced desicca-tion sensitivity as germination progresses has been demonstrated for a vari-ety of nonorthodox seed species, including Coffea arabica (Ellis and Hong,1991), Landolphia kirkii (Berjak, Pammenter, and Vertucci, 1992), Camel-lia sinensis (Berjak, Vertucci, and Pammenter, 1993), Quercus robur (Finch-Savage, Blake, and Clay, 1996), and Aesculus hippocastanum (Tompsettand Pritchard, 1998).

In general, the more heightened the state of metabolism, the greater willbe the desiccation sensitivity; this underlies not only the decreased toler-ance to water loss as germination in storage progresses, but equally, in theearlier stages of seed ontogeny desiccation damage occurs readily. Sensitiv-ity to water loss decreases with development in orthodox seeds; nonortho-dox seeds never become desiccation tolerant in the strict sense. Decreasingdesiccation sensitivity with development has been demonstrated for robustacoffee (Coffea canephora) (Hong and Ellis, 1995), for Aesculus hippo-castanum (Tompsett and Pritchard, 1993), and for Clausena lansium andLitchi chinensis (Fu et al., 1994). One of the problems posed in work on re-calcitrant seeds is that there are no outwards signs by which absolute seedmaturity can be gauged; generally, development grades virtually impercep-tibly into germination. Familiarity with individual species facilitates recog-nition of seed maturity, generally in terms of fruit developmental changes,but this lacks the precision conferred by shedding of orthodox seeds onlyafter the termination of maturation drying.

Intraseasonal Differences

Certain intraseasonal effects—which are at present largely inexplica-ble—impose degrees of variability upon seeds of individual species. Wehave consistently found that the water content of ostensibly mature seeds ofindividual species varies depending on the stage in a season at which theyare harvested (our unpublished data). Furthermore, for most species, seed-to-seed variability in the axis water content within any single harvest is sig-nificant (Berjak and Pammenter, 1997). Our other unpublished observa-tions on recalcitrant species show that fruits produced late in a season eitherwill abort or will not abscise, instead withering and dying while remainingattached to the parent. Also, late-season seeds are of very inferior quality,often showing extremely high fungal infection levels. Observations onGarcinia gummi-gutta have shown that cumulative germination values fell

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slightly as the seeds matured from cotyledon colors described as “light-cream” to “new-marigold,” and that on further darkening (to “cherry”) allgermination potential was lost (Chacko and Pillai, 1997). For Machiluskusanoi, Chien and Lin (1997) reported that the later seeds were harvested,the greater was the rate of deterioration on dehydration.

Consideration of the range of variability presented by nonorthodox seedsunderscores the difficulties in working with such seeds. No a priori assump-tions can be made about either the inherent properties or the reactions thatmight occur in response to the imposition of any particular experimental pa-rameter or set of parameters.

Seed Categorization—Discrete Behavioral Groupsor a Continuum?

The inherent variability among seeds of the broadest range of speciesand their responses to dehydration and other manipulations questionswhether seeds should be classed according to the discrete categories—orthodox, intermediate, and recalcitrant—or whether a more fluid basis ofcategorization would not be more appropriate. Although it is unquestion-ably convenient to be able to slot the seeds of individual species into a dis-crete category, this constrains investigators to use such categorizations al-though the seeds being described may not conform in all respects to thedefinitions. It is clear that there are various degrees of recalcitrant behav-ior—loosely described as maximal and minimal, and separated by a seriesof gradations (e.g., Farrant, Pammenter, and Berjak, 1988). Furthermore, ithas been shown that the rate at which recalcitrant seeds lose water deter-mines the degree of dehydration they will tolerate (Pammenter et al., 1998;Pammenter and Berjak, 1999). Similarly, seeds of all orthodox species arenot equally desiccation tolerant (Walters, 1998). Intermediate storage be-havior is taken to mean those seeds that are shed at relatively high watercontent and will withstand substantial dehydration, but not to the degree tol-erated by orthodox seeds (Hong and Ellis, 1996).

In view of the extreme variability in seed postharvest behavior which hasgradually emerged, it seems that many factors must be considered in anycategorization scheme (Berjak and Pammenter, 1994). It has been sug-gested that the postharvest behavior of seeds should be considered as con-stituting a continuum, subtended by extreme orthodoxy at the one end andthe highest degree of recalcitrance at the other, with subtle gradations be-tween the two extremes (Berjak and Pammenter, 1997, 2001).

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THE SUITE OF INTERACTING PROCESSESAND MECHANISMS INVOLVEDIN DESICCATION TOLERANCE

Many factors have been implicated in the acquisition and maintenance ofseed desiccation tolerance, and assuredly, the list is not yet complete. Whileindividual processes and mechanisms have enjoyed and fallen from favor asthe factor facilitating desiccation tolerance, it has become apparent that or-thodox behavior is the outcome of the complete expression of a suite of in-teracting mechanisms and processes. As such, acquisition and maintenanceof the desiccated state in seeds must be the outcome of coordinated multi-genic control.

In orthodox seeds profound changes accompany the acquisition of desic-cation tolerance during development and the ability to survive in the desic-cated state when mature. In the case of nonorthodox seeds the expressionand interaction of the factors concerned is incomplete (Pammenter andBerjak, 1999). Recalcitrant behavior is thus inevitably a product of seed de-velopment.

Intracellular Physical Characteristics

Desiccation-tolerant plant cells must withstand the physical strains thataccompany the volume reduction associated with the loss of considerableportions of cellular water. The water in fluid-filled spaces is generally re-placed by space-occupying insoluble material, generally protein in the vac-uoles and starch and/or lipids external to the vacuole (reviewed by Vertucciand Farrant, 1995). The cytoskeleton must have the ability to dissociate inan orderly manner, and, although there is no direct evidence, it is also possi-ble that the cell walls are plastic and can readily fold. In addition, althoughdirect data are lacking, the chromatin must assume a conformation that willprotect the integrity of the genome in the desiccated condition (Osborne andBroubiak, 1994). The nucleoskeleton, which determines nuclear architec-ture, must be modified in a strictly controlled manner.

These characteristics develop prior to or concomitant with maturationdrying in orthodox seeds but are lacking or only partially manifested in re-calcitrant seeds. Even from the relatively few studies that have been done,there appears to be a correlation between the degree of recalcitrance and themanifestation of some of these features. For example, in Avicennia marina,Ekebergia capensis, and Aesculus hippocastanum there is a decreasing de-gree of vacuolation in embryo cells and an decreasing sensitivity to dehy-dration under similar conditions (Farrant et al., 1997; Pammenter et al.,

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1998). Nevertheless, A. hippocastanum seeds are desiccation sensitive, in-dicating that although protection against physical strains is necessary, it isnot adequate in itself in conferring desiccation tolerance.

In parentheses, it should be noted that some of the ultrastructural “dam-age” observed on drying recalcitrant seeds, such as the withdrawal of theplasmalemma from the cell wall, may in fact be an artifact of fixing partiallydry tissue in an aqueous medium (Wesley-Smith, 2001).

Intracellular Dedifferentiation and Metabolic “Switch Off ”

Intracellular dedifferentiation accompanies the onset of maturation dry-ing in orthodox seeds. Mitochondria and plastids lose internal structure, andendomembranes such as the rough endoplasmic reticulum (ER) becomesubstantially reduced and the cisternae of Golgi bodies disassociated (Bainand Mercer, 1966; Klein and Pollock, 1968; Hallam, 1972). These changesimply minimization of metabolic activity, including respiration and mem-brane synthesis and processing. The reduction in membrane surface areaalso reduces the sites that would undergo substantial, often deleterious,changes upon desiccation. A further indication of metabolic “switch off” isthe cessation of DNA replication and the arrest of most embryo cells in theG1 phase (prereplication; DNA in the undoubled 2C form) with the onset ofmaturation drying (Brunori, 1967). Dehydrated orthodox seeds are effec-tively ametabolic, not simply because no water is available, but because of acontrolled shutdown of activity and dismantling of structures preceding oraccompanying maturation drying.

Marked dedifferentiation does not occur in any of the recalcitrant em-bryos of a variety of species examined to date, and respiration rates remainhigh. Interestingly, although the mitochondria of the recalcitrant embryosof both Avicennia marina and Aesculus hippocastanum remain highly dif-ferentiated, the mitochondria occupy a greater proportion of cell area in themore desiccation-sensitive species, A. marina (Farrant et al., 1997). Welack sufficient data on cell cycling in recalcitrant embryos to comment un-equivocally. However, in A. marina seeds only the most transient cessationof DNA replication occurs, with resumption of DNA synthesis (the S phase)resulting in its entering the more vulnerable doubled state (4C) soon aftershedding and the DNA of newly shed seeds is severely damaged by only aslight degree of dehydration (Boubriak et al., 2000). In the temperate recal-citrant species Acer pseudoplatanus, on the other hand, more than 60 per-cent of the embryo cells have been reported to be arrested in the 2C state,although this might be associated with the dormancy of these seeds (Finch-Savage et al., 1998).

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Free Radicals, Reactive Oxygen Species,and Antioxidant Systems

Free radicals, which include reactive oxygen species (ROS), are strongoxidants and can cause, inter alia, peroxidation of membrane lipids leadingto impairment of membrane structure and function. Reactive oxygen spe-cies are naturally produced during normal metabolism, but tissues contain arange of both enzymatic and nonenzymatic antioxidants which function toprevent injurious consequences of “escaped” ROS. However, if metabolismis disturbed (by, for example, dehydration) there is the potential for unregu-lated ROS production with deleterious consequences such as oxidation ofmacromolecules and membrane deterioration. To prevent damage duringthe early stages of maturation drying, the presence and optimal operation ofantioxidants is essential.

In developing recalcitrant seeds metabolism is not programmed to be“switched off”; it continues, if not unabated, certainly at a relatively highlevel. It appears that although recalcitrant seeds/embryos do possess anti-oxidants (and there are variations among species), these may become im-paired or otherwise unable to cope with the level of ROS generation accom-panying slow dehydration (Hendry et al., 1992; Finch-Savage et al., 1993;Côme and Corbineau, 1996; Tommasi, Paciolla, and Arrigoni, 1999).

The Presence and Operation of Putatively Protective Molecules

Sucrose with Certain Oligosaccharides or Sugar Alcohols

Maturing orthodox seeds accumulate considerable amounts of sucroseand oligosaccharides (usually raffinose and/or stachyose) (Koster and Leopold,1988), or sucrose coaccumulates with galactosyl cyclitols (Obendorf, 1997),depending on the species. As dehydration proceeds, these mixtures form ahighly viscous supersaturated solution known as a glass. This glassy (vitri-fied) state can become so viscous as to curtail molecular diffusion (Wil-liams and Leopold, 1989; Koster 1991; Leopold, Sun, and Bernal-Lugo,1994; Bryant, Koster, and Wolfe, 2001), thus minimizing unregulated me-tabolism and its deleterious consequences. It is possible that the life span ofmature orthodox seeds under defined storage conditions is influenced bythe stability of the glassy state (Leopold, Sun, and Bernal-Lugo, 1994). Be-cause they are metastable, glasses tend to break down, and this phenome-non may underlie the inevitable deterioration of orthodox seeds during stor-age.

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Interestingly, the recalcitrant seeds of several species do accumulate su-crose and oligosaccharides (Farrant, Pammenter, and Berjak, 1993; Finch-Savage and Blake, 1994; Lin and Huang, 1994; Steadman, Pritchard, andDey, 1996), which may be present in mass ratios conducive to glass forma-tion (Horbowicz and Obendorf, 1994). However, vitrification would occuronly at water contents lower than those at which recalcitrant seeds lose via-bility on slow drying (whether naturally after being shed or experimentallyafter harvest).

Late Embryogenic Accumulating/Abundant Proteins (LEAs)

Synthesis of the set of robust, hydrophilic proteins termed LEAs (dehy-drin-like proteins) precedes maturation drying during orthodox seed devel-opment (Galau, Hughes, and Dure, 1986; Kermode, 1990). Convincing evi-dence indicates that LEAs are somehow involved in the acquisition andmaintenance of desiccation tolerance in orthodox seeds, perhaps becausetheir amphipathic nature facilitates interaction with a wide range of macro-molecules and ions, thus preventing denaturation of the macromoleculesunder dehydrating conditions (Blackman, Obendorf, and Leopold, 1995;Close, 1997).

The situation with respect to the occurrence and possible role of LEAs inrecalcitrant seeds is equivocal. Such proteins occur in recalcitrant seeds of avariety of species, from grasses to trees, from a range of habitats (Finch-Savage, Pramanik, and Bewley, 1994; Gee, Probert, and Coomber, 1994;Farrant et al., 1996). On the other hand, LEAs were conspicuously absentfrom recalcitrant seeds of ten tropical wetland species tested (Farrant et al.,1996). Similarly, although seed small heat-shock proteins have been impli-cated in desiccation tolerance, a homologous protein isolated from the coty-ledons of recalcitrant Castanea sativa seeds obviously does not confer tol-erance (Collada et al., 1997).

The evidence of the occurrence of both appropriate sugar/oligosac-charide combinations and LEAs in recalcitrant seeds underscores the con-tention that no one factor can be considered to be the key factor in either theacquisition or the maintenance of desiccation tolerance. The phenomenonmust be the result of the interplay of a variety of mechanisms and processeswhich will surely emerge as being under multigenic control.

Amphipathic Substances

It has been suggested that endogenous amphipathic substances may par-tition into membrane lipid bilayers during dehydration, preventing the for-

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mation of the gel phase in the desiccated state (Hoekstra et al., 1997;Golovina, Hoekstra, and Hemminga, 1998). On rehydration, the amphi-paths have been shown to partition back into the cytoplasm. Although thismay be a further mechanism involved in desiccation tolerance in orthodoxseeds, the status of amphipathic molecules in recalcitrant seeds has not yetbeen ascertained.

The Ability for Damage Repair on Rehydration

Storage of orthodox seeds at high temperatures and water contentscauses damage that decreases vigor and brings about viability loss. How-ever, before viability is reduced, the decreased vigor is manifested as an in-creasing time lag between seed imbibition and radicle extension. Duringthis period, intracellular repair mechanisms become operational and repairmust be effected before germination can occur (e.g., Osborne, 1983). Re-pair during this lag phase in orthodox seeds occurs at the level of proteinmacromolecules (Mudgett, Lowensen, and Clarke, 1997), membranes (Berjakand Villiers, 1972), and nucleic acids (Elder et al., 1987). In fact, the effi-cacy of osmopriming of low-vigor orthodox seeds is because repair pro-cesses occur while the seeds are held at water potentials that allow this me-tabolism but preclude germination (Bray, 1995).

There are very few studies of repair by damaged recalcitrant seeds. How-ever, following rehydration of the highly recalcitrant seeds of Avicenniamarina, no DNA repair is possible once 22 percent of the originally presentwater has been lost, suggesting a very inadequate DNA repair system com-pared with orthodox seeds (Boubriak et al., 2000). In terms of free-radicalscavenging processes, evidence suggests that antioxidant systems fail dur-ing dehydration of desiccation-sensitive seeds and seedlings and are as-sumed to remain ineffective on rehydration. It appears that the repair mech-anisms of recalcitrant seeds are as sensitive to water loss as all otherprocesses.

The mechanisms and processes outlined constitute some of a suite ofprotective mechanisms, probably all of which must be present, that act to-gether to confer tolerance to dehydration and the ability to survive for ex-tended periods in the dry state. However, the list is probably by no meanscomplete, with essential developmental phenomena remaining to be identi-fied. In seeds that are not orthodox the features are represented to differingextents, and some may not be present at all. This may be the underlyingcause of the differing degrees of recalcitrance observed among species.

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DRYING RATE AND CAUSES OF DAMAGEIN RECALCITRANT SEEDS

It is now well established that the response of recalcitrant seeds, or axesexcised from seeds, to drying depends on the rate at which water is lost(Normah, Chin, and Hor, 1986; Pammenter, Vertucci, and Berjak, 1991;Pammenter et al., 1998; Pritchard, 1991; Kundu and Kachari, 2000; Pottsand Lumpkin, 2000). Although in a few exceptions drying rates intermedi-ate between slow and rapid appear to favor survival to relatively low watercontents (e.g., cacao, Liang and Sun, 2000; Warburgia salutaris, Kiokoet al., 1999), seeds or axes that are dried very rapidly can survive to the low-est water contents, most probably because insufficient time is allowed forthe accumulation of damage that occurs when the material is dried slowly.However, no matter how fast the water loss, recalcitrant material cannot bedried to as low a water content as orthodox seeds; there is an absolute lowerlimit below which recalcitrant seeds will not survive. These data have beeninterpreted as suggesting at least two types of damage can occur on dryingdesiccation-sensitive seeds. At higher water contents (above the lowerlimit) aqueous-based degradative oxidative processes, initiated because ofdisturbance of ongoing metabolism, lead to the accumulation of damage.This kills the seeds if drying is slow, but if dehydration is sufficiently rapidthis damage does not accumulate to lethal levels. However, if material issubjected to rapid drying, at lower water contents (below the lower limit) di-rect damage consequent upon removing water from membrane and macro-molecular surfaces occurs virtually instantaneously and rapidly kills the tis-sue. These types of damage have been referred to as “metabolism-deriveddamage” and “desiccation damage sensu stricto,” respectively (Pammenteret al., 1998; Walters et al., 2001).

Whatever the details of the primary events initiating metabolism-linkeddamage as a consequence of water stress, opinion generally favors oxida-tive processes to be a major cause of lethal damage. In particular, evidencethat uncontrolled generation of reactive oxygen species occurs, leading ulti-mately to peroxidation, has been linked to observable or measurable mem-brane damage and cell death (Hendry et al., 1992; Finch-Savage et al.,1993; Finch-Savage, Blake, and Clay, 1996; Chaitanya and Naithani, 1994;Li and Sun, 1999; Leprince et al., 2000). During dehydration of the highlyrecalcitrant seeds of Avicennia marina, although data indicate responsestypical of oxidative stress, these seem to be eclipsed by catastrophic physi-cal damage (Greggains et al., 2001).

There are marked differences in the desiccation sensitivity of recalcitrantseeds of different species, when dehydrated under identical conditions,

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which are due to inherent properties of the seeds themselves (e.g., Farrant,Pammenter, and Berjak, 1989). However, the drying rate (Pammenter,Vertucci, and Berjak, 1991; Pammenter et al., 1998; Berjak, Vertucci, andPammenter, 1993), and probably also the temperature under which dehy-dration occurs (Kovach and Bradford, 1992; Vertucci et al., 1995; Ntuli et al.,1997) and the maturation status of the seeds (Berjak, Pammenter, andVertucci, 1992; Berjak, Vertucci, and Pammenter, 1993; Vertucci et al.,1994, 1995) are related to the water content at which damage occurs or is le-thal. It is thus obvious that one cannot define unqualified “critical watercontents” at which viability will be lost without specifying parameters re-lating both to the seeds/axes and the experimental conditions (Pammenteret al., 1998; Pammenter and Berjak, 1999).

The Practicalities of Handling Recalcitrantand Other Nonorthodox Seeds

The natural tendency among those who harvest seeds has long been tospread these out to dry before placing them into whatever storage facility islocally used. When sophisticated drying rooms are not available, seed dry-ing is often done by equilibration with the ambient RH, generally in theshade. This has been the common practice, as is described, for example, byKioko, Albrecht, and Uncovsky (1993) for seeds from pulpy fruits inKenya. Inevitably seeds not (immediately) recognized as being recalcitrantsustain lethal damage during this very slow method of drying, resulting incomplete non-availability of planting stocks. It is highly probable that as aresult of such practices worldwide, species producing recalcitrant seedswould have been propagated vegetatively whenever possible. For example,propagation by layering in the humid tropics might have arisen as an almostnatural consequence of plant interactions with the environment for treessuch as Mangifera indica (mango), Litchi chinensis (lychee), Durio zibeth-inus (durian), Nephelium lappaceum (rambutan), Euphoria longan (longan),and Artocarpus altilis (breadfruit) (Hartmann et al., 1997). The case ofbreadfruit is historically interesting for another reason, but also to do withthe short-lived, recalcitrant seeds: one of the contributing factors to the mu-tiny on the Bounty was that established young plants of this species, whichwere being carried from Tahiti for cultivation in the British West Indies, de-prived the sailors of a significant proportion of the ship’s water supply!

Almost without exception, the crop species used in agriculture produceorthodox seeds. It is tempting to speculate that the common crop speciescultivated worldwide were domesticated because they were useful, and thefact that the seeds could be stored greatly facilitated their domestication.

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This would have been important not only for maintaining planting stocksfrom one season to the next but also because of the maintenance of qualityof the seeds as food and feed. Furthermore, most of the domesticated cropscultivated globally have temperate origins, where species producing nonor-thodox seeds are uncommon. One exception is Zizania palustris, NorthAmerican wild rice, which is a commercially grown, aquatic species thatproduces seeds recognized to be recalcitrant (Probert and Brierley, 1989;Probert and Longley, 1989; Vertucci et al., 1995). Under natural conditions,however, the deeply dormant caryopses are shed into water, where theyoverwinter and cannot dry out. Nevertheless, long-term storage of the sub-merged seeds at low temperatures, which essentially constitutes dormancy-breaking stratification, is not an option, and thus developing methods forconservation of the seeds or excised embryonic axes of Zizania spp. hasbeen the subject of considerable research. Those investigations consideredvarious aspects of the responses to water loss, which were found to differphysiologically, ultrastructurally, and biochemically depending on the tem-perature during dehydration (Kovach and Bradford, 1992; Berjak et al.,1994; Ntuli et al., 1997), with the deleterious effects being increasingly se-vere with declining temperature. The most telling studies on Zizania spp.,however, were carried out by Vertucci and colleagues (1995) who reportedthat although critical water contents for desiccation damage under defineddehydration conditions varied with developmental status and temperature,all equated to a common water activity value (aw) of 0.90. Those authors, inpresenting a model interrelating this water activity value with water contentand temperature, proposed that optimum storage conditions could be pre-dicted for caryopses of Zizania spp. from different populations, and thatlong-term conservation should be possible at –20°C. Since then, Touchelland Walters (2000) have employed the optimal drying conditions eluci-dated for Zizania palustris embryos and have achieved success in theircryopreservation.

Caryopses of Zizania spp. probably proved amenable to cultivation inthe first place because of their dormancy and aquatic habitat, and there areother dormant, recalcitrant seeds (e.g., Wasabia japonica, Nakamura andSathiyamoorthy, 1990; Aesculus hippocastanum, Pritchard et al., 1999), al-though away from the temperate regions, dormancy seldom seems to coex-ist with recalcitrance. Some informally grown “vegetable” species in thetropics produce recalcitrant seeds. One such example is Telfairia occiden-talis, an annual cucurbit native to tropical Africa, which provides a popularleaf and stem vegetable (Akoroda, 1986). However, as the seeds are edibleand a source of oil, as well as being extremely short-lived when dehydrated,planting stock is scarcely available. Successful cultivation of T. occidentalisand species producing similar seeds will probably rely heavily on methods

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of vegetative propagation and/or germplasm cryopreservation, if these canbe developed.

Whether one is considering the tropics or the temperate zones, the phe-nomenon of seed recalcitrance (or indeed, of any degree of nonorthodox be-havior) is predominantly a characteristic of tree species. Hence the handlingof such seeds is more properly in the province of silviculture or horticulturethan of agriculture—although it needs to be borne in mind that thousands ofthousands of species of unknown seed behavior might prove immenselyvaluable as cultivated crops in the future.

Harvest and Transport Criteria

Because recalcitrant seeds are hydrated and metabolically active, it is vi-tal that they are harvested in the best possible condition that will optimizenot only any storage period that might be necessitated, but also germinationperformance if planted immediately. Unless such seeds are enclosed infleshy or impermeable fruits, they are liable to start losing water as soon asthey are shed. This necessitates daily harvests throughout the season. Evenwhen immediate dehydration does not occur, the physiological status of theseeds changes, more or less rapidly depending on the species, as a conse-quence of their ongoing development or progress into germination, whichin turn influences their desiccation sensitivity (e.g., Farrant, Pammenter,and Berjak, 1986; Berjak, Farrant, and Pammenter, 1989; Berjak, Pam-menter, and Vertucci, 1992; Berjak, Vertucci, and Pammenter, 1993; Finch-Savage, Blake, and Clay, 1996; Tompsett and Pritchard, 1998), and this isalso the case for seeds categorized as intermediate (e.g., Ellis and Hong,1991). An additional, very significant problem in collecting fruits or seedsfrom the ground is that they will have been exposed to a spectrum of micro-organisms in addition to any they already harbored before being shed. Forexample, in the case of acorns, their infection by the aggressive pathogenSclerotinia pseudotuberosa Rehm. (Ciboria batschiana) occurs only as aconsequence of their contact with the ground (Kehr and Schroeder, 1996).For all these reasons, it is preferable to harvest fruits or seeds directly fromthe parent plant. However, this practice is not without other problems. Be-cause recalcitrant and other nonorthodox, seeds do not undergo maturationdrying, there are few clear indications of the state of maturity of the seeds.Outward criteria such as fruit color or, e.g., in the case of Avicennia marina,how readily the structure will detach from the peduncle are used, but theseare approximate rather than definite indicators of seed maturity. Intra- andinterseasonal variability in seed properties add to the difficulties of predict-ing their postharvest responses. Nevertheless, harvesting directly from the

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parent plant remains the procedure of choice, as this practice avoids themore serious problems of seed dehydration, increasing desiccation sensitiv-ity, and further infection.

For these same reasons, it is essential that the seeds are transported underoptimal conditions to the central facility, whether this be a repository or alaboratory. The means and duration of transport will determine what pre-cautions need to be taken for nonorthodox seeds. If it is merely a matter oftransporting the seeds rapidly over a short distance, then it is sufficient toenclose them in plastic bags or other containers that will not permit waterloss. However, when long intervals intervene between collection and deliv-ery of seeds, then further precautions are necessary. When possible, trans-port of seeds within the fruits is best, despite the fact that this necessitatesgreater volumes and weights of the consignments. If this is impossible, thenthe seeds themselves need to be treated with fungicide before packaging, tominimize fungal proliferation in the high humidity conditions of the con-tainers necessary to prevent seed water loss. However, as surface applica-tions of fungicide are not effective in curtailing the activity of mycelium be-low the pericarp/testa, recourse to seed treatments with systemic fungicides(our unpublished data) may be necessary. Obviously, however, the efficacyof such fungicides in curtailing proliferation of the specific fungi involved,as well as prior establishment that they cause no seed damage, is necessary.The effects of the seed-associated fungi need to be eliminated, or at leastminimized, for two major reasons, the first being the obvious deteriorationof the seeds by fungal degradation and toxin production. The second pointis that fungal respiration produces metabolic water; thus, even if the fungithemselves are relatively benign, the seeds are provided with an additionalsource of water and consequently are likely to become more metabolicallyactive. This, in turn, could result in earlier radicle emergence than wouldotherwise occur in hydrated storage, rendering the resultant seedlings use-less for storage and of dubious value as planting stock.

The fact that recalcitrant and other nonorthodox seeds types are activelymetabolic and require transportation in closed containers to minimize waterloss imposes the problem of the storage atmosphere becoming anoxic(Smith, 1995). To counteract this, periodically containers need to be openedbriefly and the seeds mixed around. As an alternative to closed containers,transport of recalcitrant seeds may be in moist medium, as described byKioko, Albrecht, and Uncovsky (1993). Those authors advocate the use ofsawdust, peat, vermiculite, or sand as providing a suitable medium, whichalso has been the practice in Kenya for moist storage of such seeds. Use ofmoist-medium packaging for transport does, however, lend further bulk andweight to the consignment. Nevertheless, convenience may have to be for-feited, as the objective is to transport the seeds under the best possible con-

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ditions for vigor and viability retention, as well as to minimize any (addi-tional) infection. Although more difficult to achieve, temperature duringtransport of recalcitrant seeds should be as low as can be managed, but notlow enough to damage those that are chilling sensitive.

If transport of recalcitrant seeds over long distances—whether enclosedwithin the fruits or not—is to be most efficient, then the time factor needs tobe minimized. Thus, at least for experimental purposes, consignment by airfreight is generally used. Under these conditions additional precautionsneed to be taken. First, one needs to be sure that the fruits or seeds arestowed in the temperature- and pressure-controlled hold of the aircraft, of-ten loosely described as the “live animal” hold. This is because at high alti-tudes hold temperature can be so low that freezing damage to the seedsmight occur, especially if the flight duration is several hours. Second, it isimperative that the packages are not labelled as “perishable material,” asthis inevitably will result in the consignment being held under refrigeratedconditions, especially prior to the flight and on receipt at the destination.Third is the matter of the international rules governing plant and seed im-portation and quarantine if the material is to be sent from one country to an-other. It is essential that the appropriate documentation accompanies theconsignment, which means that the receiver must have arranged for an im-portation permit that must be valid for the date of receipt of the fruits orseeds and that the exporter has provided the necessary phytosanitary certifi-cate for the species concerned. Although it may seem simple in theory tofulfill these conditions, in practice there are frequently difficulties, with theloss of precious material as a result of a inappropriate handling or substan-tial delays. We have found the cost of employing the services of a reliableinternational firm of couriers to be well worthwhile in minimizing the trou-ble and losses that can otherwise be incurred.

In some cases—particularly when seeds are very short-lived and/or col-lecting missions are protracted and transport difficulties acute—in vitrocollecting techniques can be employed (Pence, 1996; Engelmann, 1997).Englemann (1997) describes in vitro field collection of coconut and cacaoto involve extraction of embryonic axes (in a plug of endosperm for thesespecies), surface sterilization of plugs with calcium hypochlorite or com-mercial bleach, dissection out of the embryos under a makeshift “hood,”and their inoculation onto semisolid medium. As an alternative, Engelmanncites transport of the endosperm plugs to the laboratory before embryo exci-sion and notes the efficiency of such in vitro field collecting if suitable pre-cautions are taken. In these cases, however, whether the material is to bestored or not, germination followed by initial onward growth needs to beunder in vitro conditions, as will be discussed as follows. It should be noted

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that similar field collection technology applied to vegetative propagatorymaterial can be very successful.

Short- to Medium-Term Storage

To be successful, any storage regime must ensure that seeds retain unim-paired (or only minimally impaired) vigor and viability for a practicallyuseful time period, which means from harvest until they are required forplanting. In these terms, storage of recalcitrant seeds in the short to mediumterm is fraught with difficulties. Examination of the data collected by Kingand Roberts (1980) shows that some two decades ago moist-stored recalci-trant seeds were recorded as retaining viability for periods from days toweeks for tropical types and months to, at most, two and one-half years, fortemperate species, with lowered temperature being an important parameterparticularly for the latter. With few exceptions, for those species for whichviability was recorded it was not high—and certainly would not satisfy thestringent requirements of the international seed trade. In addition, little wasrecorded regarding the vigor of the surviving seeds. Seeds of Q. robur wererecorded from data collated by King and Roberts (1980) as being among thelongest surviving of temperate recalcitrant types. However, it has been ourexperience (unpublished data) that Q. robur seeds obtained from an impec-cable source after they had been stored for a few months, although still via-ble, were extremely debilitated and almost all were fungally infected.

In general, some progress has recently been made in extending the stor-age longevity of recalcitrant seeds, and it has emerged that not only theirinitial quality but also the prestorage manipulation will have a marked influ-ence on the success of their short- to medium-term storage. An a priori re-quirement for viability retention of stored recalcitrant seeds is the mainte-nance of high tissue water content, as was discussed previously. This isgenerally achieved in a variety of ways. The Kenya Forestry Research Insti-tute (KEFRI) advocates the use of moisture-retaining packing media (as forseed transport), with sand noted to be the least effective because of its po-rosity (Kioko, Albrecht, and Uncovsky, 1993). Twice the volume of peat,sawdust, vermiculite, or sand to seeds is used, moistened with distilled ordeionized water, thus lessening the chances of microorganisms being intro-duced, and storage temperature is kept as low as will not cause chillingdamage. There are, however, problems even with this relatively straightfor-ward procedure, including the necessity of keeping the seeds aerated byturning the mixture regularly, which could cause mechanical damage andmust increase the chances of microorganisms being introduced. In addition,the periodic remoistening of the packing medium may be necessary, with

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the amount of water used being carefully controlled, otherwise the seedwater content will increase with the concomitant speeding up of the germi-nation processes. In this regard, for storage of Camellia sinensis seedsBhattacharya, Rahman, and Basu (1994) have suggested use of moist sandincubation with polyethylene glycol (PEG) to maintain the water potentialat –0.5 MPa, together with the use of 0.05 percent mercuric chloride.

The storage containers used in Kenya vary from glass to plastic jarswhich may be rubber lined, with tightly closing lids, or heavy-gauge poly-thene bags (Kioko, Albrecht, and Uncovsky, 1993). Those authors recordnonorthodox seeds of a spectrum of chilling-tolerant species to survive forup to a year at 1 to 4°C when stored in moist medium. Other investigatorshave reported using similar means for short- to medium-term storage of re-calcitrant seeds. For example, high-quality seeds of Inga uruguensis har-vested directly from the tree maintained 80 percent viability for up to 80 dwhen cold stored in moist vermiculite, but germinated in storage within 20to 30 d when maintained at ambient temperature (Barbedo and Cicero,2000). Those authors reported that inclusion of abscisic acid (ABA) in themoistening water improved the storage capacity, especially of immatureseeds. The success of ABA application in extending storage longevity of re-calcitrant seeds is not likely to be universal, as this will depend not only onmaturity status but also on whether seeds of particular species are respon-sive to this growth regulator. In the case of the highly recalcitrant, chilling-sensitive (our unpublished data) seeds of Avicennia marina, inclusion ofABA in encapsulating gel designed to take the place of the pericarp had nobeneficial effects in extending storage longevity in addition to those af-forded by the gel alone (Pammenter, Motete, and Berjak, 1997).

Other authors, too, have reported marked success in extending storagelongevity of recalcitrant or otherwise nonorthodox seeds, even those thatare chilling sensitive. For Aporusa lindleyana, Kumar, Thomas, and Push-pangadan (1996) report that seeds that lose viability within a few days inslowly dehydrating conditions retained better than 90 percent viability afterone year of storage in airtight polycarbonate bottles maintained constantlyat 30°C or at a range between 20/30°C, although time taken to germinate in-creased by an order of magnitude, indicating a substantial loss of vigor.However, the data of Kumar, Thomas, and Pushpangadan (1996) indicatethat the A. lindleyana seeds selected for storage must have been of highquality at the outset.

Achievement of a useful storage period for hydrated, recalcitrant seeds(wet or hydrated storage) depends critically on the quality of the seeds atharvest, which includes their infection status. In terms of inherent seedquality, Trichilia dregeana seeds of poor quality (as a result of a presumedheat stress after shedding) when wet stored declined in viability from 100

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percent to about 20 percent over three weeks at 16°C, and to 0 percentwithin two weeks at 25°C (Drew, Pammenter, and Berjak, 2000). In con-trast, high-quality, uninfected seeds of the same species have retained via-bility for eight months and longer (our unpublished observations).

In our laboratory, where seeds are stored for experimental purposes, wegenerally use plastic buckets with sealing lids. The clean containers aresterilized with sodium hypochlorite before use, after which distilled waterto a depth of about 10 to 20 mm is introduced. A wick of sterile paper towel-ing is used to line the lower section of the bucket wall, in order to achieveand retain a saturated atmosphere. The size of the bucket is chosen to com-plement the number and dimensions of the seeds, which are placed (usuallyin a monolayer) on a grid suspended over the water in the base of the bucket.The seeds themselves will have been surface sterilized before storage, blot-ted dry with sterile paper towel, and usually dusted with a benomyl-basedfungicide. Our recent (as yet unpublished) studies have indicated that an ad-ditional prestorage step of great value to storage of the seeds of several spe-cies is treatment with a systemic fungicide “cocktail.” As an alternative touse of the buckets, similarly pretreated seeds of some species have beenfound to survive well in polythene bags. This has the advantage of a farmore efficient use of space in the seed store, as bulky buckets take up a lot ofspace. The storage temperatures used in our laboratory are either 16 ± 2°C,for seeds suspected to be chilling sensitive, or 6 ± 2°C. Interestingly, al-though Smith (1995) cautions against allowing anoxic conditions to buildup, we have seldom encountered problems with hydrated seeds stored inpolythene bags, despite the limited airspace.

Whatever the precautions taken for hydrated storage of recalcitrantseeds, retention of vigor and viability for the longest period possible (whichvaries greatly depending on the species) depends critically not only on ini-tial seed quality, but also the elimination—or at least the minimization—ofthe associated mycoflora. In early work on Avicennia marina seeds, it wasnoted that as the intact propagules became increasingly dehydrated, theybecame more and more resistant to water uptake when reimbibed (Berjak,Dini, and Pammenter, 1984). We later became aware that it was not that thelarge embryo would not take up water, but that a thick, interwoven mat offungal mycelium—which after desiccation appeared to have become hy-drophobic—had insidiously replaced the pericarp. In an attempt to elimi-nate the source of peripheral inoculum, we removed the pericarp from freshseeds and, to retain tissue water, encapsulated them in a crude, slightlyacidic, potassium alginate gel. This study, aimed at investigating aspects ofmetabolism and ultrastructure during hydrated storage of A. marina seeds,produced surprising results in the highly significant extension of their stor-age longevity (Pammenter, Motete, and Berjak, 1997; Motete et al., 1997).

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Although gel encapsulation did not appear to offer any metabolic advan-tages to the seeds, compared with the decoated condition (pericarp re-moved, but not encapsulated), the efficacy of the gel as a fungistatic treat-ment suggested itself. The beneficial effects of similar gel encapsulation onstorage longevity of recalcitrant seeds of several species have, however,been equivocal; possibly this is because, as the preparation is a crude ex-tract, its properties are not consistent. Nevertheless, our current studies haveshown the freshly prepared alginate gel to be strongly fungicidal and, atleast in the case of seeds of A. marina, Artocarpus integer, and Trichiliadregeana, to extend hydrated storage life span.

Work on A. marina seeds involving an aerosol application of fungicide toseeds after pericarp removal and surface sterilization and periodically dur-ing storage has shown clearly that when the effects of the mycoflora are cur-tailed in this way, the longevity of the hydrated seeds is significantlyextended (Calistru et al., 2000). Coupled with this were the observationsthat subcellular integrity was maintained while deteriorative ultrastructuralchanges within the tissues were associated with the presence of fungalstructures on the surfaces of cotyledons and axis in seeds that had been in-oculated after surface sterilization. As a result of these observations, and onthe basis that all recalcitrant seeds are metabolically active and those ofmany species can be considered to be developing seedlings, work is nowproceeding on the evaluation of systemic fungicidal cocktails to be used aspretreatments before hydrated storage of recalcitrant seeds. The unpub-lished results show for several species that storage longevity can be signifi-cantly extended. However, it would be entirely incorrect to assume thatelimination of the effects of seed-associated fungi would confer indefinitestorability on recalcitrant and other nonorthodox propagules. Antifungaltreatments appear to make possible considerably more effective periods ofshort- to medium-term storage, but ultimately it is the demands of ongoinggerminative metabolism in the absence of additional water that will curtaillongevity (Pammenter et al., 1994). There is evidence that this is the resultof water-stress-related generation of highly destructive reactive oxygenspecies which will rapidly bring about deterioration and death of the seedsin hydrated storage (Hendry et al., 1992; Finch-Savage et al., 1993; Finch-Savage, Blake, and Clay, 1996; Chaitanya and Naithani, 1994; Li and Sun,1999; Leprince et al., 2000).

Clearly, if metabolic rate can be kept to a minimum by lowered storagetemperatures, the timing of the onset of the germination phase that demandsadditional water can also be postponed. This is the basis of storage at thelowest temperature that recalcitrant seeds of individual species will toler-ate. For example, Suszka and Tylkowski (1980) reported considerably in-creased storage longevity of Quercus robur seeds at around 0°C, and Prit-

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chard and colleagues (1995) have shown significant extension of viabilityof Araucaria hunsteinii seeds in cold storage where temperatures were suf-ficiently low to preclude radicle emergence. However, use of lowered tem-peratures that would be effective is precluded, especially in the case of sometropical species, e.g., Theobroma cacao, Nephelium lappaceum, Hopeaspp., and others (King and Roberts, 1980). This is vividly illustrated in thecase of Hevea brasiliensis seeds, for which Normah and Chin (1991) foundthat deterioration was slowest at 27°C and most rapid at 10°C. Another ap-proach that has been periodically suggested for prolonging storage life spanof recalcitrant seeds is to lower the water content to a point that might cur-tail microorganism proliferation and would limit metabolism to the point ofprecluding the progress of germination (e.g., Hong and Ellis, 1996); this istermed “subimbibed” storage. The water content of axes and storage tissuesof recalcitrant seeds at shedding is variable both inter- and intraseasonally,as discussed previously, but is presumably correlated with the physiologicalstatus of the seeds at that time. If improved storage longevity is observed forseeds of particular species at water contents that are somewhat lower thanmight be expected, this could well be a function of the combination of phys-iological status and water content at harvest not yet having reached its low-est level.

Nevertheless, King and Roberts (1980) recorded that although subim-bibed storage had been used for seeds of a variety of truly recalcitrant andother nonorthodox species, this did not seem to be effective even in the rela-tively short term. Since then, data have further indicated that partial dryinghas adverse consequences for seeds of tropical species (King and Roberts,1982; Corbineau and Côme, 1986a,b, 1988; Xia, Chen, and Fu, 1992) aswell as those of temperate origin (Tompsett and Pritchard, 1993; Pritchardet al., 1995). Drew, Pammenter, and Berjak (2000), working with T. dregeanashowed that for seeds of this species, lowering the water content by lessthan 15 percent was associated with gross ultrastructural deterioration thatoccurred far more rapidly than in seeds stored in parallel at the original wa-ter content. Furthermore, fungal proliferation posed a considerably greaterproblem on and in the subimbibed T. dregeana seeds. The deleterious ef-fects of subimbibed storage are interpreted in terms of our current view thatthe time for which recalcitrant seeds are held at unfavorable water contents,even if these are relatively high, greatly exacerbates metabolism-linkeddamage (Pammenter et al., 1998; Walters et al., 2001).

There is no doubt that if only good-quality seeds are selected for storage,and if these can be suitably treated to curtail—or even eliminate—the inter-nal as well as the surface-associated microflora, then hydrated storage inscrupulously clean containers held at the lowest temperature commensuratewith vigor and viability retention will achieve the longest possible useful

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life span. It is, however, essential that the stored seeds be monitored periodi-cally, so that any which show signs of deterioration, fungal growth, or visi-ble germination (i.e., radicle protrusion) can be removed from the container.At any signs of fungal proliferation, it is advisable to retreat the remainingseeds. However, it must be appreciated that hydrated storage offers only aninterim measure to conserve planting resources of recalcitrant seeds.

Seedling Slow Growth As a Means of Storage

Although traditionally slow growth storage is used for material culturedin vitro, including embryogenic callus, plantlets, shoot apices, etc. (Engel-mann, 1997), there is also the possibility of maintaining seedlings undergrowth-restricting conditions, instead of contending with the difficulties in-herent in short- to medium-term storage of recalcitrant or other nonortho-dox seeds (Chin, 1996). Chin records survival for nine months of seedlingsof Shorea sp., maintained at 16°C and 70 percent RH, with an 8 h low-lightphotoperiod, while those of Calamus sp. (rattan palm) could be maintainedfor two years. As suggested by Chin (1996), seedling slow growth in shadedconditions under natural canopies is an inexpensive alternative, based onconditions in forests where light limitation restricts seedling growth until agap occurs.

Cryostorage

On the basis that hydrated storage of intact seeds is essentially only ashort-term option, other methodology must be employed to ensure the con-servation of the genetic resources of species producing recalcitrant—orother nonorthodox—seeds. From our current viewpoint, it appears thatcryostorage is the only option. This involves storage of the material at tem-peratures between –80 and –196°C, using ultra-deep-freeze facilities or liq-uid nitrogen, respectively. However, to cryostore hydrated, metabolicallyactive material in such a way that lethal damage is obviated requires consid-erable manipulation both before and after the storage phase.

It is obvious that the intact seed would be the best form of the germplasmto cryostore, as theoretically it should be merely a matter of thawing thepropagules after storage and planting them out. However, it is impossible tofreeze seeds at the high water contents characteristic of the newly shed orharvested condition, as cooling would be slow and accompanied by lethalice formation. In addition, the great majority of recalcitrant seeds cannot bedried sufficiently rapidly to maintain viability at the low water contents thatare required to prevent ice crystal formation on freezing. Even if this were

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possible, they are generally just too large to cool (freeze) successfully.However, cryostorage of intact seeds has been achieved for isolated specieswhere the propagules are small enough and dry sufficiently rapidly to em-ploy this technological approach. This is the case for Azadirachta indica(neem), a species for which seed postharvest behavior appears to vary fromorthodox through intermediate to recalcitrant, perhaps as a function ofprovenance (Chaudhury and Chandel, 1991; Berjak and Dumet, 1996).

Neem seeds, previously shown to be chilling sensitive and to exhibitnonorthodox (tending to recalcitrant) behavior (Berjak et al., 1995) wereable to be dried to very low water contents within 1 to 2 d when maintainedon a layer of activated silica gel (Berjak and Dumet, 1996). The exo- andmesocarp had been removed prior to desiccation, as retention of these cov-erings was associated with lethally slow dehydration. After cooling at an in-termediate rate in cryotubes plunged into liquid nitrogen, 70 to 75 percentof the endocarp-enclosed seeds had retained viability when sampled overthe four months that they were stored in this cryogen (Berjak and Dumet,1996). Whole seeds of Wasabia japonica have also survived cryopreserva-tion in liquid nitrogen after rapid dehydration to low water content (Pottsand Lumpkin, 2000), and, similarly, those authors found that slow dehydra-tion was deleterious. Hu, Guo, and Shi (1993) cryopreserved seeds of tea(Camellia sinensis) after dehydration to low water content and documentedthat equilibration on a low-moisture-content sand bed after retrieval fromliquid nitrogen favored germination. In the studies described the seeds sur-vived desiccation to 0.16 g·g–1 (Azadirachta indica), 0.17 g·g–1 (Wasabiajaponica), and 0.16 g·g–1 (Camellia sinensis). As has been shown by Wes-ley-Smith et al. (1992), it is only at such low water contents that the speci-mens will survive the relatively slow cooling in liquid nitrogen achieved forthe seeds in the studies outlined. This presupposes that the tissues of nonor-thodox seeds of individual species will withstand such drastic dehydrationand retain viability at ambient temperature (i.e., before introduction intoliquid nitrogen) even for very short periods. It is also significant that two ofthese species, W. japonica and C. sinensis, are not of tropical provenance, asit seems that generally temperate provenance is correlated with the abilityof seeds/seed organs to withstand—even temporarily—such extreme dehy-dration.

However, this generalization is not without exception. Kioko and col-leagues (1999) dehydrated seeds of the tropical species Warburgia salutarisrelatively rapidly to about 0.1 g·g–1. In that study, 30 percent of the seedssurvived cryostorage after relatively slow cooling in liquid nitrogen, and insubsequent experiments (unpublished), healthy saplings have been pro-duced from 65 percent of cryostored seeds of the related species W. ugan-densis. Slow dehydration is invariably lethal for seeds of Warburgia spp.,

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thus providing an additional example of the practical value of rapid dryingin retaining viability of nonorthodox seeds to low water contents. Viabilityretention at low water contents of seeds of Warburgia spp. is, however, onlytransient, and these seeds will die within two weeks if maintained at ambi-ent temperature or in cold storage (unpublished data). Their survival ofrapid dehydration, however, provides viable material for cryostorage.

In the studies cited previously the seeds shared two characteristics:(1) they tolerated desiccation to water contents that would be lethal for mostrecalcitrant seeds, however rapidly they could be dried, and (2) they wererelatively small. Large seeds—the norm for most recalcitrant species—cannot be rapidly dehydrated, simply because of their size. The solution tothe problem of providing a suitable form of the germplasm for cryopreser-vation for most large-seeded species is the use of excised embryonic axes(as originally demonstrated by Normah, Chin, and Hor, 1986) which can bedehydrated extremely rapidly by the process of flash drying (Berjak et al.,1990). Zygotic embryonic axes offer the same advantages to conservationof genetic diversity as do intact seeds, but their use involves several proce-dures, all of which can cause problems in achieving the ultimate objective,the production of normal, vigorous plants (e.g., Berjak et al., 1996; Engel-mann, 1997; Dumet, Berjak, and Engelmann, 1997). However, these can beovercome in most cases by careful experimentation—presently on a spe-cies-specific basis.

Excision of embryonic axes removes them from the stored nutrients vitalfor their ongoing development, imposing the necessity of germination in vi-tro on a medium supplying a carbon source and other factors. It is impera-tive that appropriate in vitro conditions be established as the first step in de-veloping a cryopreservation protocol for axes of any species. Because of theoften short fruiting season and the fact that the seeds cannot be stored effec-tively, frequently a season is lost before any further trials can be carried out.A major problem with germination of excised axes in vitro is that associatedmicroorganisms (usually fungi) must be successfully eliminated, as thesewill proliferate swiftly and vigorously while the explant is still recoveringfrom excision and other manipulations essential for cryostorage.

Fungi pose a major problem in tissue culture irrespective of the origin ofthe explants, thus their removal—and exploration of the use of antibiotics toeliminate any bacterial contaminants if necessary—are a priori require-ments for work with zygotic axes. The same is true for the production of so-matic embryos or culture of shoot apices, which offer alternate genetic re-sources for conservation by cryopreservation. When zygotic axes are usedas explants, it is imperative that they are surface sterilized after excisionfrom the seeds, which have usually been similarly treated themselves. Assurface sterilants kill microorganisms, they are potentially injurious to the

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excised axes as well; therefore, the least injurious but effective treatmentmust be identified on a species-specific basis. In general, however, the axesof most species can withstand immersion for 10 to 15 min in a 1 percent so-dium hypochlorite solution containing a few drops of a wetting agent suchas Tween 20 (Dumet, Berjak, and Engelmann, 1997). Those authors havealso reviewed cases in which, because sodium (or calcium) hypochlorite istoo harsh, ethanol or a 0.1 percent solution of mercuric chloride has beenemployed. In some isolated instances (e.g., Warburgia spp., unpublishedobservations) all surface sterilants have proved so injurious, that use of theaxes as explants is precluded. In such cases, except in isolated instances inwhich whole seeds can be cryopreserved (e.g., Warburgia spp.), use ofexplants alternative to zygotic axes, particularly somatic embryos or shootapices, generally is necessary. An additional problem posed by the seed-associated fungi is that the inoculum may be internal and, in such cases, sur-face sterilization alone will be ineffective. We are presently experimentingwith the use of solutions of systemic fungicides, on the basis that recalci-trant seeds, being hydrated and metabolic, are in many cases more likeseedlings and should assimilate these without deleterious consequences.

The water content of axes excised from fresh seeds is generally far toohigh for cryopreservation without lethal ice formation during the coolingstep. This is at least partly because the wetter the tissues, the slower thecooling rate and thus the longer the time for passage of the axis through thetemperature range that facilitates ice crystallization (Wesley-Smith, Wal-ters, et al., 2001). Thus the axes need to be dehydrated as rapidly as possibleto water contents that will not cause dehydration damage but will facilitatenoninjurious freezing. This was first achieved by placing the excised axes ina laminar flow cabinet (Hevea brasiliensis, Normah, Chin, and Hor, 1986;Araucaria hunsteinii, Pritchard and Prendergast, 1986) and has been em-ployed since (e.g., Camellia sinensis, Chaudhury, Radhamani, and Chan-del, 1991, Cocos nucifera, Assy-Bah and Engelmann, 1992; Quercus faginea,Gonzales-Benito and Perez-Ruiz, 1992; Q. robur, Poulsen, 1992). How-ever, generally more rapid and thus effective dehydration is obtained byflash drying (Berjak et al., 1990) using a relatively simple apparatus inwhich a flow of air passes through a grid from below, with the axes posi-tioned on the grid. The air is vented through holes in the lid of the small-volume container. Since then, the flash drying apparatus has been im-proved, with the use of a small fan and the introduction of activated silicagel (Wesley-Smith, Pammenter, et al., 2001). Flash drying achieves, in amatter of minutes to an hour or two, water contents that will facilitate rapidcooling (freezing).

Wesley-Smith and colleagues (Wesley-Smith et al., 1992; Wesley-Smith,Pammenter, et al., 2001) have presented data for Camellia sinensis and

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Aesculus hippocastanum showing that the more rapidly axes are cooled, thehigher is the water content at which they can be successfully frozen, andsimilar results have been obtained for Quercus robur axes (Berjak et al.,1999). The success of rapid freezing is explained as the minimization of thetime that the axes spend in the temperature range in which ice crystalliza-tion occurs during cooling to the temperature of the cryogen. The generalmeans of achieving this is by rapid introduction of unenclosed (naked) axesinto subcooled liquid nitrogen (–210°C), although cooling enclosed axes atsomewhat lower rates has been reported as successful for Hevea brasil-iensis (Normah, Chin, and Hor, 1986), Cocos nucifera (Assy-Bah andEngelmann, 1992), and Coffea spp. (Abdelnour-Esquivel, Villalobos, andEngelmann, 1992). However, cooling very rapidly appears to be the optimalprocedure for the axes of most, but perhaps not all, species (J. Wesley-Smith, personal communication). For Euphoria longan, successful cryo-preservation has been reported for axes subjected to precooling to –18°Cbefore being introduced into the cryogen (Fu, Xia, and Tang, 1993). In gen-eral, however, slow cooling will be successful only when intracellular vis-cosity has been increased by prior dehydration to very low water contents.These low water contents will either cause desiccation damage sensu stricto(Pammenter et al., 1998; Walters et al., 2001) or poise the axes perilouslyclose to the point where this will occur. Considering the various manipula-tions to which excised zygotic axes are subjected during the cryopreserva-tion protocol, application of excessive stress—even if it is non-lethal in it-self—is tantamount to predisposing the tissues to further injury duringsubsequent steps of the procedure (Berjak et al., 1999). Thus a balanceneeds to be sought—presently on a species-specific basis—between theleast extent of dehydration commensurate with the rapid cooling rate re-quired to achieve successful freezing of excised axes. So far, no mention hasbeen made of the use of cryoprotectants—which are generally osmoticathat may or may not penetrate the tissues but have in common the effect ofdecreasing water contents. It has been our experience that their use, al-though effective with somatic embryos, appears highly injurious to excisedzygotic axes (unpublished observations).

Cryopreservation in liquid nitrogen at –196°C has the potential to con-serve germplasm indefinitely, although, as occurs under any storage condi-tions, extraneous factors causing free-radical generation cannot be obviated.However, at the temperature of liquid nitrogen, metabolism is suspended, aswould be any associated deleterious reactions, and, within the reinforcedcryocontainers, there should be minimal effects of any extraneous factors. Itis when cryostored germplasm is retrieved from the cryogen, however, thatthe potential for damage again becomes a reality.

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It has long been known that to avert damage rewarming of frozen tissuesneeds to be rapid (e.g., Sakai, Otsuka, and Yoshida, 1968). When partiallyhydrated axes are retrieved from cryostorage, passage through the tempera-ture range that promotes crystallization events must be as rapid as possible,as is the case during cooling. Thus thawing is best achieved by immediateimmersion at about 40°C. Naked axes have usually been plunged into dis-tilled water around that temperature, while cryovials containing axes areimmediately introduced into a water bath. Although there is no doubt aboutthe requirement for rapid warming (thawing), we have considerable dis-quiet about plunging naked, partially dehydrated axes into distilled water,as it seems impossible that imbibitional damage and/or considerable leak-age of solutes from the partially dehydrated tissues can be avoided. How-ever, trials using liquid medium (i.e., the growth medium to be used for invitro germination, minus the gelling agent) have not been encouraging (un-published data).

Work with successfully cryopreserved zygotic axes of Quercus roburthat were warmed by plunging into distilled water showed that while rootsdeveloped strongly they showed no gravitropic response, and shoot ultra-structure showed a progression through derangement to necrosis (Berjaket al., 2000). Both abnormalities were obviated by warming the axes in a so-lution containing calcium and magnesium ions calculated to favor intra-cellular skeleton reconstitution. Analyses showed that normal shoot apicalmeristem structure and function were promoted and that statoliths devel-oped strongly in the root cap columella, where these geosensors were nota-bly absent after water thawing (Berjak et al., 2000). Following in vitro re-covery of axes, germination, and hardening off, vigorous saplings resulted.By using an interim recovery period in the dark for retrieved Zizaniapalustris axes, Touchell and Walters (2000) showed that light is another im-portant parameter that might generally impede axis recovery.

It should be realized, however, that the science of cryopreservation of zy-gotic axes is still in its formative stages, but, by painstaking species-by-spe-cies experimentation, there is a gradual emergence of general principles.However, many confounding factors still prevent success with individualspecies, factors which are presently largely unknown. Probably these arenot technological but reside in the seeds/axes themselves—in terms of themarked diversity of species producing recalcitrant and other nonorthodoxseed types—and in the significant inter- and intraseasonal variability withinany one species.

There are, however, examples of such seeds in which the zygotic axis isentirely unsuitable for cryopreservation, generally because it is far too large(Berjak et al., 1996; Dumet, Berjak, and Engelmann, 1997). In such casesalternative explants need to be developed or identified to enable conserva-

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tion of the germplasm. There are two common routes for this, namely, de-velopment of somatic embryos and use of shoot apices, both adding consid-erably to the time-consuming phases of in vitro experimentation or practice.Elaboration on the detailed methodology of either is beyond the scope ofthis chapter, for which the interested reader is directed to a recent review onsomatic embryogenesis by Ibaraki and Kurata (2001) or, for propagationfrom shoot apices (meristem tip cultures), to George (1993/1996).

Whether the genetic resources of species producing essentially unstor-able seeds are cryopreserved as shoot apices, zygotic axes, somatic em-bryos—or embryogenic callus from which these are usually produced—thepractical matter of their distribution and onward propagation has common-ality. The most straightforward way of achieving this would be despatch ofthe cryostored explants in liquid nitrogen, within specially constructeddewar containers. However, this would require the availability at the receiv-ing end of all the necessary in vitro facilities and the expertise necessary toretrieve the explants from cryostorage, thaw them without damage, andpropagate plantlets which would then need to be hardened off. These re-quirements clearly will seldom be able to be met. At the other end of thelimited spectrum of possibilities is the transport of small, hardened-offplants or, less conveniently, of in vitro plantlets, each in a sterile polythenebag. The most practical approach, if it can be achieved, would be the pro-duction of so-called artificial or synthetic seeds.

Synthetic seeds are usually produced by encapsulating propagatory ma-terial in alginate beads, in which a variety of additives may be included.This technology is most frequently employed for propagation of somaticembryos (e.g., Bajaj, 1995a,b). Shoot apices, too, may be successfully en-capsulated in alginate beads and subsequently stored, including those ofsome tropical forest trees (Maruyama et al., 1997). Elaborations of this arti-ficial seed technology are easily achieved; e.g., Patel and colleagues (2000)encapsulated plant material in a solution of carboxymethylcellulose andcalcium chloride, which maintained a liquid core within the alginate beads.

It is conceivable that a similar technology may be used for the distribu-tion of germplasm of species producing recalcitrant seeds. Although cryo-storage of already encapsulated zygotic axes or somatic embryos is possi-ble, this would exacerbate the problems of efficient cooling, as the alginatebead would necessarily be of significantly greater volume than the propa-gatory unit. Hence, encapsulation after safe retrieval from cryostorage is en-visaged as the better approach. It would be necessary that the alginate beadimposed conditions slowing ongoing germinative metabolism. Althoughuse of crude potassium alginate has achieved this for whole seeds ofAvicennia marina (Motete et al., 1997), treatment with mannitol or ABAhas been reported to retard ongoing development of recalcitrant somatic

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nucellar embryos of Mangifera indica (mango), although not in the contextof cryopreservation (Pliego Alfaro et al., 1996).

Although considerable research will be necessary, it seems possible thatartificial seeds could be produced containing zygotic axes, somatic em-bryos, or apical meristems of species producing recalcitrant seeds. Suchunits would probably have to include appropriate fungicides and antibiot-ics, as well as a nutrient source in the case of axes or somatic embryos tosustain germination if the propagatory unit is to be planted in soil, ratherthan being developed further in vitro.

Although this concluding thought is conceptual rather than reflecting acurrent reality, if the production of such artificial seeds encapsulatingpropagatory material retrieved from cryostorage can be realized, then prac-tical methods of conservation, distribution, and exchange of recalcitrant—and other nonorthodox—germplasm will have been achieved.

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Connor, K.F. and Bonner, F.T. (1996). Effects of desiccation on temperate recalci-trant seeds: Differential scanning calorimetry, gas chromatography, electron mi-croscopy, and moisture studies on Quercus nigra and Quercus alba. CanadianJournal of Forest Research 26: 1813-1821.

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Hay, F.R., Probert, R., Marro, J., and Dawson, M. (2000). Toward the ex situ con-servation of aquatic angiosperms: A review of seed storage behavior. In Black,M., Bradford, K.J., and Vázqez-Ramos, J. (Eds.), Seed Biology—Advances andApplications (pp. 161-177). Wallingford, UK: CABI Publishing.

Hendry, G.A.F., Finch-Savage, W.E., Thorpe, P.C., Atherton, N.M., Buckland,S.M., Nilsson, K.A., and Seel, W.E. (1992). Free radical processes and loss ofseed viability during desiccation in the recalcitrant species Quercus robur L.New Phytologist 122: 273-279.

Hoekstra, F.A., Wolkers, W.F., Buitink, J., Golovina, E.A., Crowe, J.H., andCrowe, L.M. (1997). Membrane stabilization in the dry state. Comparative Bio-chemistry and Physiology 117A: 335-341.

Hong, T.D. and Ellis, R.H. (1990). A comparison of maturation drying, germina-tion, and desiccation tolerance between developing seeds of Acer pseudo-platanus L. and Acer platanoides L. New Phytologist 116: 589-596.

Hong, T.D. and Ellis, R.H. (1995). Interspecific variation in seed storage behaviorwithin two genera: Coffea and Citrus. Seed Science and Technology 23: 165-181.

Hong, T.D. and Ellis, R.H. (1996). A protocol to determine seed storage behavior.In Engels, J.M.M. and Toll, J. (Eds.), IPGRI Technical Bulletin No. 1. Rome: In-ternational Plant Genetic Resources Institute.

Horbowicz, M. and Obendorf, R.L. (1994). Seed desiccation tolerance and stor-ability: Dependence on flatulence-producing oligosaccharides and cyclitols—Review and survey. Seed Science Research 4: 385-405.

Hu, J., Guo, G., and Shi, S.X. (1993). Partial drying and postthaw preconditioningimprove survival and germination of cryopreserved seeds of tea (Camelliasinensis). Plant Genetic Resources Newsletter No. 93: 1-4.

Ibaraki, Y. and Kurata, K. (2001). Automation of somatic embryo formation. PlantCell, Tissue and Organ Culture 65: 179-199.

Kehr, R.D. and Schroeder, T. (1996). Long-term storage of oak seeds—New meth-ods and mycological aspects. In Proceedings of the ISTA Tree Seed PathologyMeeting (pp. 50-61). Opocno, Czech Republic, October 1996. International SeedTesting Association.

Kermode, A.R. (1990). Regulatory mechanisms involved in the transition from seeddevelopment to germination. Critical Reviews in Plant Science 9: 155-195.

King, M.W. and Roberts, E.H. (1980). Maintenance of recalcitrant seeds in storage.In Chin, H.F., and Roberts, E.H. (Eds.), Recalcitrant Crop Seeds (pp. 53-89).Kuala Lumpur, Malaysia: Tropical Press SDN.BHD.

King, M.W. and Roberts, E.H. (1982). The imbibed storage of cocoa (Theobromacacao) seeds. Seed Science and Technology 10: 535-540.

Kioko, J., Albrecht, J., and Uncovsky, S. (1993). Seed collection and handling. InAlbrecht, J. (Ed.), Tree Seed Handbook of Kenya (pp. 34-54). Nairobi, Kenya:GTZ Forestry Seed Centre.

Kioko, J., Berjak, P., Pritchard, H., and Daws, M. (1999). Studies of postsheddingbehavior and cryopreservation of seeds of Warburgia salutaris, a highly endan-gered medicinal plant indigenous to tropical Africa. In Marzalina, M., Khoo,K.C., Jayanthi, N., Tsan, F.Y., and Krishnapillay, B. (Eds.), Recalcitrant Seeds

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(pp. 365-371). Kuala Lumpur, Malaysia: Forest Research Institute Malasya(FRIM).

Klein, S. and Pollock, B.M. (1968). Cell fine structure of developing lima beanseeds related to seed desiccation. American Journal of Botany 55: 658-672.

Koster, K.L. (1991). Glass formation and desiccation tolerance in seeds. PlantPhysiology 96: 302-304.

Koster, K.L. and Leopold, A.C. (1988). Sugars and desiccation tolerance in seeds.Plant Physiology 88: 829-832.

Kovach, D.A. and Bradford, K.J. (1992). Temperature dependence of viability anddormancy of Zizania palustris var. interior seeds stored at high moisture con-tents. Annals of Botany 69: 297-301.

Kumar, C.A., Thomas, J., and Pushpangadan, P. (1996). Storage and germination ofAporusa lindleyana (Wight) Baillon, an economically important plant of West-ern Ghats (India). Seed Science and Technology 25: 1-6.

Kumar, K.K. and Chacko, K.C. (1999). Seed characteristics and germination of a‘shola’ forest tree: Bhesa indica (Bedd.) Ding Hou. Indian Forester 125: 206-211.

Kundu, M. and Kachari, J. (2000). Desiccation sensitivity and recalcitrant behaviorof seeds of Aquilaria agallocha Roxb. Seed Science and Technology 28: 755-760.

Leopold, A.C., Sun, W.Q., and Bernal-Lugo, I. (1994). The glassy state in seeds:Analysis and function. Seed Science Research 4: 267-274.

Leprince, O., Buitink, J., and Hoekstra, F.A. (1999). Axes and cotyledons of recalci-trant seeds of Castanea sativa Mill. exhibit contrasting responses of respirationto drying in relation to desiccation sensitivity. Journal of Experimental Botany50: 1515-1524.

Leprince, O., Harren, F.J.M., Buitink, J., Alberda, M., and Hoekstra, F.A. (2000).Metabolic dysfunction and unabated respiration precede the loss of membraneintegrity during dehydration of germinating radicles. Plant Physiology 122: 597-608.

Li, C.R. and Sun, W.Q. (1999). Desiccation sensitivity and activities of free radical-scavenging enzymes in recalcitrant Theobroma cacao seeds. Seed Science Re-search 9: 209-217.

Liang, Y.H. and Sun, W.Q. (2000). Desiccation tolerance of recalcitrant Theobromacacao embryonic axes: The optimal drying rate and its physiological basis. Jour-nal of Experimental Botany 51: 1911-1919.

Lin, T.-P. and Chen, M.-H. (1995). Biochemical characteristics associated with thedevelopment of the desiccation-sensitive seeds of Machilus thunbergii Sieb. andZucc. Annals of Botany 76: 381-387.

Lin, T.-P. and Huang, N.-H. (1994). The relationship between carbohydrate compo-sition of some tree seeds and their longevity. Journal of Experimental Botany 45:1289-1294.

Martins, C.C., Nakagawa, J., and Bovi, M.L.A. (2000). Desiccation tolerance offour seedlots from Euterpe edulis Mart. Seed Science and Technology 28: 101-113.

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Maruyama, E., Kinoshita, I., Ishii, K., Ohba, K., and Saito, A. (1997). Germplasmconservation of the tropical forest trees, Cedrela odorata L., Guazuma crinitaMart., and Jacaranda mimosaefolia D. Don., by shoot tip encapsulation in cal-cium-alginate and storage at 12-25°C. Plant Cell Reports 16: 393-396.

Motete, N., Pammenter, N.W., Berjak, P., and Frédéric, J.C. (1997). Response of therecalcitrant seeds of Avicennia marina to hydrated storage: Events occurring atthe root primordia. Seed Science Research 7: 169-178.

Mudgett, M.B., Lowensen, J.D., and Clarke, S. (1997). Protein repair L-isoaspartylmethyltransferase in plants: Phylogenetic distribution and the accumulation ofsubstrate proteins in aged barley seeds. Plant Physiology 115: 1481-1489.

Nakamura, S. and Sathiyamoorthy, P. (1990). Germination of Wasabia japonicaMatsum. seeds. Journal of the Japanese Society for Horticultural Science 59:573-577.

Normah, M.N. and Chin, H.F. (1991). Changes in germination, respiration rate andleachate conductivity during storage of Hevea seeds. Pertanika 14: 1-6.

Normah, M.N., Chin, H.F., and Hor, Y.L. (1986). Desiccation and cryostorage ofembryonic axes of Hevea brasiliensis Muell.-Arg. Pertanika 9: 299-303.

Normah, M.N., Ramiya, S.D., and Gintangga, M. (1997). Desiccation sensitivity ofrecalcitrant seeds—A study on tropical fruit species. Seed Science Research 7:179-183.

Ntuli, T.M., Berjak, P., Pammenter, N.W., and Smith, M.T. (1997). Effects of tem-perature on desiccation responses of seeds of Zizania palustris. Seed Science Re-search 7: 145-160.

Obendorf, R.L. (1997). Oligosaccharides and galactosyl cyclitols in seed desicca-tion tolerance. Seed Science Research 7: 63-74.

Osborne, D.J. (1983). Biochemical control of systems operating in the early hoursof germination. Canadian Journal of Botany 61: 3568-3577.

Osborne, D.J. and Boubriak, I.I. (1994). DNA and desiccation tolerance. Seed Sci-ence Research 4: 175-185.

Pammenter, N.W. and Berjak, P. (1999). A review of recalcitrant seed physiology inrelation to desiccation-tolerance mechanisms. Seed Science Research 9: 13-37.

Pammenter, N.W. and Berjak, P. (2000). Evolutionary and ecological aspects of re-calcitrant seed biology. Seed Science Research 10: 301-306.

Pammenter, N.W., Berjak, P., Farrant, J.M., Smith, M.T., and Ross, G. (1994). Whydo stored, hydrated recalcitrant seeds die? Seed Science Research 4: 187-191.

Pammenter, N.W., Greggains, V., Kioko, J.I., Wesley-Smith, J., Berjak, P., andFinch-Savage, W.E. (1998). Effects of differential drying rates on viability re-tention of recalcitrant seeds of Ekebergia capensis. Seed Science Research 8:463-471.

Pammenter, N.W., Motete, N., and Berjak, P. (1997). The response of hydrated re-calcitrant seeds to long-term storage. In Ellis, R.H., Black, M., Murdoch, A.J.,and Hong, T.D. (Eds.), Basic and Applied Aspects of Seed Biology (pp. 671-687).Dordrecht, the Netherlands: Kluwer Academic Publishers.

Pammenter, N.W., Vertucci, C.W., and Berjak, P. (1991). Homeohydrous (recalci-trant) seeds: Dehydration, the state of water and viability characteristics inLandolphia kirkii. Plant Physiology 96: 1093-1098.

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Patel, A.V., Pusch, I., Mix-Wagner, G., and Dunlop, K.D. (2000). A novel encapsu-lation technique for the production of artificial seeds. Plant Cell Reports 19:868-874.

Pence, V.C. (1996). In vitro collection (IVC) method. In Normah, M.N., Narimah,M.K., and Clyde, M.M. (Eds.), In Vitro Conservation of Plant Genetic Re-sources (pp. 181-190). Kuala Lumpur, Malaysia: Percetakan Watan Sdn.Bhd.

Pliego Alfaro, F., Litz, R.E., Moon, P.A., and Gray, D.J. (1996). Effect of abscisicacid, osmolarity and temperature on in vitro development of recalcitrant mangonucellar embryos. Plant Cell, Tissue and Organ Culture 44: 53-61.

Potts, S.E. and Lumpkin, T.A. (2000). Cryopreservation of Wasabia spp. seeds.CryoLetters 18: 185-190.

Poulsen, K. (1992). Sensitivity to low temperatures (–196°C) of embryonic axesfrom acorns of the pedunculate oak (Quercus robur L.). CryoLetters 13: 75-82.

Pritchard, H.W. (1991). Water potential and embryonic axis viability in recalcitrantseeds of Quercus rubra. Annals of Botany 67: 43-49.

Pritchard, H.W. and Prendergast, F.G. (1986). Effects of desiccation and cryopre-servation on the in vitro viability of embryos of the recalcitrant seed speciesAraucaria hunsteinii. Journal of Experimental Botany 37: 1388-1397.

Pritchard, H.W., Steadman, K.J., Nash, V.J., and Jones, C. (1999). Kinetics of dor-mancy release and the high-temperature germination response in Aesculus hip-pocastanum seeds. Journal of Experimental Botany 50: 1507-1514.

Pritchard, H.W., Tompsett, P.B., and Manger, K.R. (1996). Development of a ther-mal time model for the quantification of dormancy loss in Aesculus hippo-castanum seeds. Seed Science Research 6: 127-135.

Pritchard, H.W., Tompsett, P.B., Manger, K., and Smidt, W.J. (1995). The effect ofmoisture content on the low temperature responses of Araucaria hunsteinii seedand embryos. Annals of Botany 76: 79-88.

Probert, R.J. and Brierley, E.R. (1989). Desiccation intolerance in seeds of Zizaniapalustris is not related to developmental age or the duration of postharvest stor-age. Annals of Botany 64: 669-674.

Probert, R.J. and Longley, P.L. (1989). Recalcitrant storage physiology in threeaquatic grasses (Zizania palustris, Spartina anglica and Porteresia coarctata).Annals of Botany 63: 53-63.

Roberts, E.H. (1973). Predicting the storage life of seeds. Seed Science and Tech-nology 1: 499-514.

Sakai, A., Otsuka, K., and Yoshida, S. (1968). Mechanism of survival in plant cells atsuper-low temperatures by rapid cooling and rewarming. Cryobiology 4: 165-173.

Smith, R.D. (1995). Collecting and handling seeds in the field. In Guarino L., Rao,V.R., and Reid, R. (Eds.), Collecting Plant Genetic Diversity (pp. 419-456).Wallingford, UK: CAB International.

Steadman, K.J., Pritchard, H.W., and Dey, P.M. (1996). Tissue-specific solublesugars in seeds as indicators of storage category. Annals of Botany 77: 667-674.

Suszka, B. and Tylkowski, T. (1980). Storage of acorns of the English oak (Quercusrobur L.) over 1-5 winters. Arboretum Kórnickie 25: 199-229.

Tommasi, E., Paciolla, C., and Arrigoni, O. (1999) The ascorbate system in recalci-trant and orthodox seeds. Physiologia Plantarum 105: 193-198.

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Tompsett, P.B. (1992). A review of the literature on storage of dipterocarp seeds.Seed Science and Technology 20: 251-267.

Tompsett, P.B. and Pritchard, H.W. (1993). Water changes during development inrelation to the germination and desiccation tolerance of Aesculus hippocastanumL. seeds. Annals of Botany 71: 107-116.

Tompsett, P.B. and Pritchard, H.W. (1998). The effect of chilling and moisture sta-tus on the germination, desiccation tolerance and longevity of Aesculus hippo-castanum L. seed. Annals of Botany 82: 249-261.

Touchell, D. and Walters, C. (2000). Recovery of embryos of Zizania palustris fol-lowing exposure to liquid nitrogen. CryoLetters 21: 261-270.

Vertucci, C.W., Crane, J., Porter, R.A., and Oelke, E.A. (1994). Physical propertiesof water in Zizania embryos in relation to maturity status, water content and tem-perature. Seed Science Research 4: 211-224.

Vertucci, C.W., Crane, J., Porter, R.A., and Oelke, E.A. (1995). Survival of Zizaniaembryos in relation to water content, temperature and maturity status. Seed Sci-ence Research 5: 31-40.

Vertucci, C.W. and Farrant, J.M. (1995). Acquisition and loss of desiccation toler-ance. In Kigel, J. and Galili, G. (Eds.), Seed Development and Germination(pp. 237-271). New York: Marcel Dekker, Inc.

von Teichman, I. and van Wyk, A.E. (1994). Structural aspects and trends in theevolution of recalcitrant seeds in the dicotyledons. Seed Science Research 4:225-239.

Walters, C.W. (1998). Ultra-dry seed storage. Seed Science Research 8 (Supple-ment no. 1).

Walters, C.W., Pammenter, N.W., Berjak, P., and Crane, J. (2001). Desiccationdamage, accelerated ageing and respiration in desiccation tolerant and sensitiveseeds. Seed Science Research 11: 135-148.

Wesley-Smith, J. (2001). Freeze-substitution of dehydrated plant tissues: Artefactsof aqueous fixation revisited. Protoplasma 218: 154-167.

Wesley-Smith, J., Pammenter, N.W., Berjak, P., and Walters, C. (2001). The effectsof two drying rates on the desiccation tolerance of embryonic axes of recalcitrantjackfruit (Artocarpus heterophyllus Lamk.) seeds. Annals of Botany 88: 653-664.

Wesley-Smith, J., Vertucci, C.W., Berjak, P., Pammenter, N.W., and Crane, J.(1992). Cryopreservation of recalcitrant axes of Camellia sinensis in relation todehydration, freezing rate and thermal properties of tissue water. Journal ofPlant Physiology 140: 596-604.

Wesley-Smith, J., Walters, C., Pammenter, N.W., and Berjak, P. (2001). Interac-tions among water content, rapid (nonequilibrium) cooling to -196°C, and sur-vival of embryonic axes of Aesculus hippocastanum L. seeds. Cryobiology 42:196-206.

Williams, R.J. and Leopold, A.C. (1989). The glassy state in corn embryos. PlantPhysiology 89: 977-981.

Xia, Q.H., Chen, R.Z., and Fu, J.R. (1992). Moist storage of lychee (Litchi sinensisSonn.) and longan (Euphoria longan Steud.) seeds. Seed Science and Technol-ogy 20: 269-279.

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SECTION IV:INDUSTRIAL QUALITY OF SEEDS

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Chapter 11

Processing Quality Requirements for Wheat and Other Cereal GrainsProcessing Quality Requirements for Wheatand Other Cereal Grains

Colin W. WrigleyFerenc Bekes

INTRODUCTION

A seed is a plant’s means of producing another plant, thereby perpetuat-ing the species. The requirements for a seed to fulfill this role are for it toprovide a good store of nutrients to supply the new plant in the early stagesof its growth. Safe storage of these nutrients is essential throughout what-ever conditions may occur until the right combination of moisture and tem-perature triggers the germination response. The ability of plants to provideseeds as stores of nutrients has also made them an attractive food source forhumankind. Because grains have been recognized as an important foodsince prehistory, the first propagation of seed-bearing plants was an impor-tant phase in the development of humankind, marking the transition fromhunter-gatherer to a settled agricultural existence. These developments led,in turn, to the building of permanent dwellings and to a wide range of cul-tural activities.

THE RANGE OF GRAIN SPECIES USED INDUSTRIALLY

Thousands of years of such agricultural practice have led to the selectionof species that suit humankind’s requirements. Further improvements haveled to the development of cultivated varieties within those species withquality attributes that are even better suited to processing requirements. Ashort list of such species (Table 11.1) includes members of both monocoty-ledonous and dicotyledonous plants. The success of this approach to ob-taining food is indicated by the worldwide cultivation of billions of seed-bearing plants annually. This activity yields well over 2 billion tonnes of

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grain of all species (counting only the production of those countries thatregister relevant statistics) (Wright, 2001). As a result, grains are the mainsource of protein and energy for humankind, either directly by processingthe seeds into food or indirectly by the ingestion of animal foods (meat,milk, eggs) following the feeding of grains to animals.

TABLE 11.1. Major grain species used for human food and for animal feed

Family Common name Genus and speciesMonocotyledons

Gramineae (Pooids) Bread wheat Triticum aestivumDurum wheat Triticum durumTriticale Triticosecale speciesRye Secale cerealeBarley Hordeum vulgareTritordeum Hybrid between Hordeum

chilense and durum wheatBambusoids Oats Avena sativa

Rice Oryza sativaWild rice Zizania aquatica

Eu-panicoids Pearl millet Pennisetum glaucumFinger millet Eleusine coracanaJapanese millet Echinochloa species

Andropogonoids Sorghum Sorghum bicolorMaize (corn) Zea mays

DicotyledonsAmaranthaceae Amaranth Amaranthus speciesPolygonaceae Buckwheat Fagopyrum esculentumMalvaceae Cottonseed Gossypium speciesLeguminosae Lupin Lupinus species

Pea Pisum sativumPeanut Arachis hypogaeaSoybean Glycine max

Cruciferae Rapeseed Brassica napusLinaceae Linseed Linum usitatissimumCompositae Sunflower Helianthus annuus

Source: Adapted from Watson and Wrigley, 1984, and from Wrigley and Bekes,2001.

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Dicotyledonous Grains

The taxonomic diversity of grains is indicated in Table 11.1. The greatestdiversity is found among the dicots, including various grain legumes (alsocalled pulses) (including lupins, peas, peanuts), soybeans, and oilseedssuch as rapeseed/canola, safflower, linseed/linola, sunflower, and cotton-seed. Collectively, oilseed production totals about 300 million tonnes annu-ally, with most of this (250 million tonnes) being crushed for oil production(Wright, 2001). The utilization of oilseeds is reviewed in Chapter 12. Worldproduction of pulses totals nearly 60 million tonnes, with about 25 percentof this being produced in India. The dicot group of grains also contains arange of less common grains, such as amaranth, for which valuable modesof utilization have been developed (Lehmann, 1996).

Monocotyledonous Grains—The Cereals

On the other hand, the range of monocot grains all belong to a singlefamily of grassy plants (the Gramineae), being grouped under the general ti-tle “cereals” (Watson and Wrigley, 1984). Wheat is unique in its ability toform a viscoelastic dough after milling and mixing with water. Many of therange of wheat-based products in Figure 11.1 are generally familiar, espe-cially the conventional leavened bread. Less familiar to Westerners are thevarious flat (Arabic) breads in Figure 11.1, the wide range of pasta and noo-dle types in Figure 11.2, and the Chinese steamed bread in Figure 11.3.However, all these wheaten products rely on the gluten-forming proteins ofwheat. For this reason, wheat is the cereal grain for which markets are themost conscious of quality specifications. Wheat is thus the main topic ofthis chapter on quality requirements. World production of wheat totalsabout 600 million tonnes annually, coming from some 220 million hectares.World trade in wheat is about 100 million tonnes annually, the main tradingnations being the United States, Canada, Australia, Argentina, and thecountries of Europe. Wheat’s close relative, rye, is minor by comparison;world rye production is about 22 million tonnes annually, the main produc-tion regions being in eastern parts of Europe, especially Poland, Germany,and Russia (Bushuk, 2001a,b).

Rice, too, has a unique place in the human diet, providing the major(sometimes, almost the sole) source of energy and protein for many cul-tures. World production rivals that of wheat, based on the volume of paddyrice (hulled). After milling to dehulled rice, world production is about 400million tonnes. Maize fulfills a similar but distinct role for other cultures, inaddition to being the major grain used in industrial processing. Its annual

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production (approaching 600 million tonnes) is included in the statisticians’term “coarse grains,” which total 900 million tonnes annually, coveringover 300 million hectares of agricultural production. World trade in coarsegrain is about 100 million tonnes. This term also includes barley (about 150million tonnes annually), sorghum (50 million tonnes), and oats (30 milliontonnes) (Wright, 2001). Close behind wheat in having exacting quality re-quirements comes barley, with its unique ability to provide malt for brew-ing, as is reviewed in Chapter 13.

CEREAL GRAINS AND OUR DIET

Most forms of processing involve the application of moist heat togelatinize starch (making it more readily digested), to denature proteins,and to inactivate antinutritional compounds (especially for some dicotgrains). Heat processing may follow various forms of milling to remove

FIGURE 11.1. Baked goods made from wheat flour include Arabic-style flatbreads as well as the leavened square loaf of bread.

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outer husks or bran layers, thereby making the grain product more readilydigestible. Grain-based foods therefore occur almost universally in the hu-man diet, although in many different forms. However, for some individuals,cereal grains may cause dietary problems. One of the best characterized ofthese intolerances is celiac disease, a condition caused by the ingestion ofwheat gluten protein and analogous grain proteins of rye, triticale, barley,and sometimes oats (Skerritt, Devery, and Hill, 1990; Kasarda, 2001). How-ever, Table 11.1 shows that buckwheat is a distant relative of wheat (despiteits misleading name), so buckwheat is not toxic to celiacs (Skerritt, 1986).Test kits have been developed for home use to detect the presence of glutenin foods to assist celiacs in diet control (Skerritt and Hill, 1991).

Whole Grain and Fiber

On the other hand, health benefits are provided by the inclusion of theouter layers of the cereal grains in whole-grain foods. This is because theseparts of the grain (Figure 11.4) provide various vitamins and minerals, andbecause they contribute increased amounts of fiber to the diet (Malkki,2001). The cereal grains offer a wide range of high-fiber ingredients in the

FIGURE 11.2. The diversity of noodle types made from wheat

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human diet, depending on the source of the fiber and the manner of its pro-cessing, as is reviewed by Nelson (2001). A further report (American Asso-ciation of Cereal Chemists [AACC], 2001) reviews methods of defining anddetermining dietary fiber, as well as providing a list of commercially avail-able sources of fiber from the various cereals.

The correspondence between milling fractions and grain morphology isillustrated in Figure 11.4 for the wheat grain. The positions of these tissuesin a sectioned grain are shown diagrammatically in Figure 11.5. These dia-grams illustrate that white flour consists primarily of the endosperm of thegrain. Normal milling practice provides about 75 percent of the grain aswhite flour, but in some commercial operations the extraction rate may ex-ceed 80 percent. For some special products, such as Japanese udon noodles,lower extraction rates are adopted (about 60 percent). Figure 11.6 showshow the range of nutrients changes with the extraction rate used in flourmilling. Complex carbohydrates (and therefore energy) are little affected byextraction rate, and protein content is only slightly altered. However, the con-

FIGURE 11.3. Chinese steamed bread, shown by Sidi Huang of BRI AustraliaLtd. (Sydney). This type of bread is steam cooked, so that it does not develop thebrown crust normally associated with baked products.

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tents of fiber, vitamins, and minerals are progressively reduced as the flouris primarily made up of endosperm with less of the germ and bran layers.

Feed Grains

For feed uses of grains, fiber content has a distinctly different role, de-pending on whether the target animal is a ruminant (cows, horses) or not(pigs, chickens). Ruminants can utilize much of the fiber that cannot con-tribute to weight gain for nonruminants. Furthermore, there are antinutri-tional aspects of nonstarch polysaccharides (e.g., pentosans) in the diets ofnonruminants, such as chickens (Choct and Annison, 1990). However, forall feed uses of grains, there is the universal need for the essential amino ac-ids (relating to protein quality) and the energy-contributing components ofstarch and especially lipids. Suitability for use as animal feed also dependson naturally occurring antinutritional factors (enzyme inhibitors) and con-taminants, such as mycotoxins and pesticide residues. A systematic ap-proach has been proposed for determining the suitability of grain lots foranimals, depending on the composition of the grain and the specific re-quirements of the animals to be fed (Morris and Rose, 1996).

Tissues

Fruit = pericarp

Caryopsis(kernel)

Seed

Testa

Nucellar layer

Germ

Grinding fractions

Bran

Short

Flour

Germ

cuticleepicarpcross cell layertube cell layer

cuticleepicarpcross cell layertube cell layer

EpidermisEndosperm

Aleurone layerStarchy endosperm

Epiblast

Scutelum

Embryo

FIGURE 11.4. Correspondence between the morphological features of thewheat grain and the fractions that result from milling

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Pericarp

Outer pericarp

Inner pericarpThin-walled cells(remnants)Cross cellsTube cells

CreaseCavity

Cavity

(occasional)

(occasional)

Pigment strand

Prismatic endosperm

Central endosperm

{ {{EpidermisHypodermis

TestaNucellar epidermis(remnants)AleuroneSubaleurone

FIGURE 11.5. Diagrams of the transversally sectioned cereal grain (applicableto wheat, rye, or triticale), specifying the outer layers of bran and aleurone cells(Source: From W. K. Heneen and K. Brismar, 1987, Scanning electron micros-copy of mature grains of rye, wheat, and triticale with emphasis on grain shrivel-ing, Hereditas 107: 147-162. Reproduced with permission.)

Nut

rient

cont

ent

(%of

the

orig

inal

leve

l)

Extraction rate (%)

1. Carbohydrate2. Energy3. Protein4. Fat5. Minerals6. Vitamins7. Fiber

100 80 60 40

20

40

60

80

100

76

5

4

3

2

1

FIGURE 11.6. Progressive changes in the content of various nutrients in wheatflour as the milling extraction rate falls

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USES OF CEREAL GRAINS

Wheat

The great diversity of food uses of wheat (Table 11.2) includes the manytypes of breads (leavened and unleavened), flat breads (Arabic/pocket types),pizza crust, tortillas, Chinese steamed breads, noodles of many types,breakfast cereals and porridge, cakes, biscuits/cookies, scones, muffins,chapatis, extruded snack foods, and the wide range of pasta (Faridi andFaubion, 1995). Some of these baked products are illustrated in Figures11.1, 11.2, and 11.3. Flat breads are made in many types in Middle Easterncountries, in Turkey and surrounding countries, in southeastern Europe, andin the Indian subcontinent (Qarooni, Ponte, and Posner, 1992; Qarooni,

TABLE 11.2. Quality attributes preferred in wheats for specific products. In allcases, good milling is required, giving a high yield of white flour.

ProductProtein content

(%)Grain

hardnessDough

strengthBreads

Pan bread >13 Hard StrongFlat bread 11-13 Hard MediumChinese steamed bread

Northern style 11-13 Hard Medium/strongSouthern style 10-12 Medium/hard MediumGuangdong stylea 9-11 Medium/hard MediumGuangdong styleb 9 Soft/medium Weak/medium

Noodlesc

Alkaline 11-13 Hard MediumWhite 10-12 Medium/soft MediumInstant 11-12 Medium Medium

Biscuit/cake 8-10 Very soft WeakPasta >13 Very hard Very strongStarch/gluten manufactured >13 Hard

(soft preferred)Strong

Source: Adapted from Wrigley, 1994, and updated by S. Huang.aFat-containing formula (popular in Taiwan)bFormula without fatcNull-4A genotypes preferred for starch propertiesdGenotypes preferred should have soft grain and high proportions of large (Atype) starch granules

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1996). These types include lavash, barbari, taftoon, sangak, baladi, pita,tanoor, and chapati, each having distinctive characteristics and a particularshape and texture to suit a variety of ethnic origins and preferences.

A wide range of noodle types are shown in Figure 11.2. Traditionally,noodles are made by cutting a sheet of dough into strips, the dough beingmade from a hexaploid wheat (Triticum aestivum in Table 11.1). Noodlesmay be white, cream, or yellow, as well as commonly being colored by ad-ditions such as buckwheat and spinach. Alkaline noodles have a yellowcolor from the incorporation of alkaline salts, e.g., sodium carbonate or bi-carbonate (Moss, Miskelly, and Moss, 1986). In contrast to noodles, pastasare traditionally made by extruding a dry dough. Pasta products includemacaroni, spaghetti, and many other geometric forms. Pasta dough is tradi-tionally made from durum wheat—a tetraploid species that differs geneti-cally from the more common hexaploid bread wheat (Table 11.1). Never-theless, hexaploid wheat is sometimes blended with durum wheat for pastaproduction. In addition, there are the many pastry uses of wheat, includingpies, fancy baked goods, grocery flour for many home-cooking uses, and,finally, animal feed and nonfood industrial uses.

A major industrial use of wheat flour is the separation of gluten andstarch by the water washing of dough. The resulting starch goes into manyfood applications, especially those requiring thickening agents, and into awide range of industrial uses, including adhesives. The resulting gluten hastraditionally been dried and added to bread to increase dough strength andto facilitate the production of specialty products such as pizza crust andhigh-bran and fiber-increased breads. This is still the major use of vital drygluten, but dry gluten is being increasingly used as a general food additive,finding its way into breakfast cereals, cheese, processed meats, snacks, andeven chewing gum, as well as being formulated into fish and meat analogs.For nonhuman consumption, gluten is used in pet foods, animal feed pel-lets, and aquaculture feeds. Table 11.3 lists a wide range of innovative ex-tensions of gluten utilization, being used “as is” and after various forms ofmodification (Bietz and Lookhart, 1996).

Most of these wheat-based foods are dependent on the unique dough-forming quality of wheat flour and the resulting ability of dough to retainthe gas cells produced by yeast fermentation, leading to the familiar lighttexture of leavened bread. In other cases, the dough-forming quality isneeded to permit machining or hand kneading followed by sheeting andcutting (for noodles) or stretching and covering (for many pastry goods).The strength and extensibility requirements differ for the various products,as listed for the range of products in Table 11.2. Dough-forming propertiesalso depend on protein content, which is a prominent attribute determiningmarket value. Grain hardness is also a critical attribute determining process-

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ing suitability, particularly the stage of milling into white flour. For hardwheats, the crushing motion of milling causes the rupture of the starch gran-ules, causing the damaged starch to be more accessible to amylase actionduring fermentation. By contrast, the starch granules of soft wheats are re-leased intact in the milling process, with the result that their covering mem-brane acts as a protective surface, suiting them better for biscuit/cookiemanufacture and to washing out pure starch in starch-gluten production.

Rye and Triticale

Rye and triticale are the only cereals that approach the bread-makingability of wheat, but even so, they have relatively poor capabilities in that re-spect. Bread made solely from rye is generally poorly risen and very dark incolor (Bushuk, 2001a,b). In fact, the manufacture of rye bread often in-

TABLE 11.3. Novel products made from wheat gluten

Product Manner of use of glutenFilms Gluten-based packaging films and coatings can be excel-

lent edible, renewable, and biodegradable air barriers withgood mechanical properties.

Coatings Gluten coatings may protect flavor and shelf life of foods.Chemically modified gluten may have superior propertiesas paper coatings.

Polymers/resins Modified gluten hydrolyzates give flexibility and elasticity tocertain polymers and resins. Gluten, as well as starch, canbe grafted into polymers.

Inks Adding gluten to water-thinned inks can reduce drying ofpen tips, while speeding drying on some surfaces.

Laundry detergents Modified protein hydrolyzates may stabilize enzymesadded to detergents to remove stains.

Cosmetics and haircare products

Gluten hydrolyzates act as moisturizers in cosmetics andas foaming agents and conditioners in hair care products.

Adhesives Modified gluten hydrolyzates are useful in pressure-sensi-tive adhesives.

Rubber products Modified cereal flours can reinforce certain types ofnontire rubber.

Milk replacers Partially hydrolyzed wheat protein has much potential as amilk replacer in animal nutrition.

Functional-foodproducts

Acid or enzymatic hydrolysis of gluten can improve itsemulsifying, foaming, and solubility properties for use infoods.

Source: Adapted from Bietz and Lookhart, 1996.

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volves the blending of a high proportion of wheat flour to improve loafquality. Nevertheless, rye bread is a popular item in some cultures, espe-cially in eastern Europe, the site of the greatest rye production. In additionto the use of rye for bread making, it is used to a limited extent for whiskeymanufacture. Rye is well suited to cool, temperate regions and to high alti-tudes, even for poor soils. It has the agronomic advantage that the youngplants can be grazed, with adequate grain production still being assured atmaturity. The grain, however, is not well suited for animal feed, especially ifthere is contamination with ergot, which is a particular problem with rye.

The man-made hybrid triticale is an attempt to combine the agronomicadvantages of rye with wheat. However, the production of triticale is notgreat compared to the wider range of cereals. Some varieties of triticale of-fer the advantage of better baking quality than rye alone, while providingsome of the flavor characteristics of rye. The higher fiber content of triticale(compared to wheat) has been offered as a health advantage that should beconsidered for food use. In addition, claims have been made for the dietaryuse of triticale to reduce the risks of cancer and coronory heart disease(Slavin, Marquart, and Jacobs, 2000).

Barley

The premium use of barley is for malting and brewing, for which theremay be specific segregation and marketing of individual varieties sepa-rately from others, because of the unique malting properties that are pre-ferred by the maltster for one particular variety. This important use of barleyis described in detail in Chapter 13 and also in dedicated monographs suchas MacGregor and Bhatty (1993). The other major use of barley is as animalfeed—both for ruminants and nonruminants. Smaller proportions of thebarley crop are used for a range of human foods, mainly in the pearled form,resulting from abrasion to remove the outer lemma, palea, and bran layers.A novel cereal grain developed from barley is the man-made hybrid tritor-deum, an amphiploid between Hordeum chilense and durum wheat (Martinet al., 1999). Tritordeum is morphologically and agronomically similar towheat, and it shows promise of providing satisfactory baking quality.

Oats

Oats may suffer to some extent from the famous altercation between theEnglishman Samuel Johnson and the Scot Boswell; the former describingoats as a grain fit only for horses in England, although eaten by men in Scot-land. Boswell’s retort was, “And pray, where do we see such horses as were

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produced in England and such men as were reared in Scotland.” In most oat-growing countries, oats are mainly destined for animal feeding, often at thesite of production, with about 20 percent of production going for humanconsumption. Webster’s (1986) monograph is still a general source book onoats; it has been partly updated (Webster, 1996). These are complementedby a monograph on oat bran (Wood, 1993), produced to provide a reasonedsource of information at a time when extreme nutritional claims were beingmade for oat bran.

A major use of oats is as a hot breakfast cereal in the form of porridge,produced from rolled oats. The rolling process is applied to the whole groat.Heat processing is essential to inactivate enzymes (lipase, lipoxygenase,and peroxidase), which would otherwise produce soapy bitter flavors. Fur-ther processing may involve sectioning the rolled groat into smaller piecesto permit quicker cooking. Oat flakes may also be incorporated into coldbreakfast cereals, such as muesli, and into baked goods, such as muffins andcookies. Oatmeal and oat flour are major components of many infant foods.

Most genotypes of oats, as harvested, have the outer husks attached, sothese must be removed during processing for human food. There are somehull-less oat genotypes. The dehulled grain is termed the “groat.” Its aleur-one layer, surrounding the starchy endosperm, is a good source of solubledietary fiber in the form of beta-glucan. The cell walls of the endosperm arealso rich in beta-glucans. Groats have relatively high levels of protein(range of 10 to 20 percent) and fat (5 to 10 percent), compared to othercereal grains. The balance of oleic and linoleic acids is a desirable blend forhuman nutrition. The effects of genotype and growth condition on fat com-position have been studied using near-infrared spectroscopy (Krishnanet al., 2000).

The protein of oats has a high nutritional value, based on its content ofessential amino acids, relative to other cereal grains, because oats have alower content of the prolamin class of proteins and relatively more of theglobulin class of proteins (Figure 11.7). The prolamins of oats, termed“avenins,” are polymorphic—as is seen by SDS-gel electrophoresis or two-dimensional isoelectric-focusing electrophoresis. The alpha-avenins havehigher isoelectric points than the gamma avenins. The amino-acid se-quences are known for many of the avenins (Egorov et al., 1994; Shewry,1999).

Oats (and also rice) are unusual among the cereal grains in that their ma-jor protein class is the globulins, rather than the prolamins (Figure 11.7).Globulins account for about 75 percent of the seed protein content (Shot-well, 1999). The oat-globulin protein is a hexamer with subunits of about55,000 Daltons, which each comprise disulfide-linked polypeptides of32,000 and 23,000 Daltons. Oat globulin is rich in glutamine and aspar-

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agine, consistent with the role of storing nitrogen. However, oat-globulinprotein is slightly deficient in the sulfur-containing amino acids cysteineand methionine, being similar to the globulins of the legumes in this re-spect. Oat globulin shares about 70 percent amino acid sequence similaritywith the storage glutelin of rice, despite the differences in solubility of thesetwo classes of protein (Shotwell, 1999).

Rice

The production and consumption of rice are mainly concentrated in thecountries of Asia, the home of about 60 percent of the world’s population.In Bangladesh, Cambodia, Indonesia, Laos, Myanmar, Thailand, and Viet-nam, rice provides 55 to 80 percent of energy in the diet. Worldwide, riceprovides about 20 percent of the total food calories consumed (Athwal,1971). International rice trade accounts for only about 5 percent of worldproduction, and much of this trade is in specialty grades. For example, thehigh-quality scented basmati rice of Pakistan and Northern India may com-mand a fourfold price premium. The major exporters are Thailand, theUnited States, Vietnam, and Pakistan. Others include Australia, China, In-

Globulins Prolamins

7S 11/12S Other Prolamins The Prolamin Superfamily

Maize, Oats,Rice, Barley,Rye, Wheat

Wheat Oats Rice Maize Sorghum Rice Maize Oats

EmbryoAleurone Endosperm

Triticeae:

Barley, Wheat,Rye

7S Globulin Tricitin Globulin Glutelin Kafirin

-Zein -Zein

16 kDa 10 kDa 13 kDa

-Zein -Zein

Avenin

HMW S-rich S-poor

FIGURE 11.7. Classification of the cereals, according to the types of proteins intheir grains (Source: Adapted from Shewry, 1996.)

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dia, and Uruguay. Standard test procedures have been established to assessrice-cooking quality (Juliano, 1985a). The range of chemical and techno-logical aspects of rice utilization is reviewed in Juliano (1985b).

As a protein source, milled rice contains the lowest amount of protein(ca. 5 percent) among the major cereals; moreover, this protein content isnot easily digestible by humans or by monogastric animals. However, com-pared to other cereal proteins, the overall amino acid composition of riceprotein is significantly better balanced, because of its relatively high levelof lysine. The amino acid composition of rice is unusual among the cerealgrains because rice is one of the few cultivated plants in which both theglobulins and prolamins, the two classes of storage proteins of higherplants, are present in significant levels (Figure 11.7). Unlike most other cere-als, which accumulate prolamins as their primary nitrogen reserve, the ma-jor storage proteins in rice are the glutelins, which are homologous at theprimary-sequence level to the 11S globulin proteins, a class which is the domi-nant form of nitrogen deposition in legumes. Furthermore, the rice prolaminproteins have a number of characteristics that are different from the pro-lamins of most other cereals.

Based on the classic study of Betchel and Juliano (1980), the most im-portant characteristics of the deposition of nitrogen in the rice kernel arethree kinds of protein bodies in the rice endosperm, namely, large sphericalprotein bodies, small spherical ones, and crystalline protein bodies, each ofthem surrounded by a single continuous-unit membrane. The spherical pro-tein bodies form within vacuoles, but the proteins are synthesized in theendoplasmatic reticulum and in the Golgi apparatus before being trans-ported to the vacuoles by vesicles.

Removal of the husk from rough rice yields the kernel, which composesthe pericarp, seed coat, the aleurone layer, the endosperm, and the germ;this form is known as brown rice (with a protein content of 9 to 10 percent).Brown rice has a significantly higher nutritional value than white polished(milled) rice, which is the most commonly utilized rice product (with a pro-tein content of about 8 percent). The milling process for rice results in 40 to55 percent white milled rice and three major by-products: husks (20 per-cent), bran (10 percent), and “brokens” (10 to 22 percent), with protein con-tents of 3, 17, and 8.5 percent, respectively. The aleurone layer, the tissuewith the highest levels of protein and nutritionally important minor compo-nents, is removed during the process of rice milling.

The distribution of the protein fractions from the Osborne procedure offractionation for brown or white rice (Table 11.4) reflects the observationthat the levels of the albumin and globulin classes are significantly higher inthe outer layers of the seed; they decrease toward the center of the grainwhile the proportion of glutelins has an inverse distribution (Bechtel and

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Pomeranz, 1980; Takaiwa, Ogawa, and Okita, 2000). The subaleurone re-gion plays very significant role nutritionally; it is several cell layers thickand is rich in the globulin class of proteins. Its lysine content is much higherthan the proteins located in the endosperm. It is therefore desirable to millas lightly as possible to retain most of the subaleurone layer on white rice.

The albumin fraction isolated from rice is highly heterogeneous and con-tains many biologically important components. It can be separated into foursubfractions, based on the molecular size of the protein components, whichrange from 10 to 200 kDa. More than 50 individual polypeptides have beenobserved in the albumin fraction, based on fractionation by isoelectric fo-cusing. Detailed studies on many of these components have shown that ricealbumins have mostly enzymic or enzyme-inhibitor activities. Compared towheat, rye, and barley, rice contains significantly lower amounts of thehigh-pI -amylases and much higher levels of the low pI -amylases.

Maize (Corn)

Corn, indigenous to North America, was developed by Central Americannatives many centuries before Columbus arrived. Corn was the foundationof the extensive North and South American ancient civilizations and wasimportant in the agriculture and nutrition of more recent American Indianpopulations in a unique form of treatment, lime cooking. This form of pro-cessing is still widely used today, with its nutritional advantages, in themaking of corn-based products such as tacos and tortillas (Serna-Salvidar,Gomez, and Rooney, 1990). Columbus carried corn seed to Europe, whereit became established as an important crop in southern latitudes. Neverthe-less, U.S. corn production accounts for over half the total world productionand 80 percent of the annual world corn exports (see monograph by Watsonand Ramsrad, 1987).

In countries where corn is an important crop, it is the principal compo-nent of livestock feeds and most of it is fed to farm animals. In only a fewcountries is corn a major constituent of human diets. In developed coun-tries, corn is consumed mainly as popcorn, sweet corn, corn snacks, and oc-

TABLE 11.4. The distribution of protein classes for brown and white rice accord-ing to the Osborne fractionation method

Albumin Globulin Prolamin GlutelinBrown rice 10.8 9.7 2.2 77.3White rice 6.5 12.7 8.9 71.9

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casionally as corn bread. About one-third of processed corn is used to pro-duce corn starch, sweeteners, corn oil, and various feed by-products. Theremainder is utilized to prepare various food products and alcoholic bever-ages. Beyond alkali treatment, corn is prepared in several ways as humanfood: (1) parched to be eaten whole; (2) ground to make hominy, corn meal,or corn flour; and (3) converted to a variety of breakfast foods (Hoseney,1986).

The corn kernel is the largest of all cereals. Kernels are usually flatteneddue to the pressure from adjacent kernels during growth. Dent maize is themost widely grown type of maize. The structure of the kernel is made up offour principal parts: (1) the epidermis and the seed coat (in practice calledthe “hull” or “bran”), (2) the endosperm (including the aleurone layer),(3) the germ, and (4) the tip cap. This last part is the point of attachment ofthe cob to the plant. It may or may not stay with the kernel during shelling.Table 11.5 shows typical values for the chemical composition of the variousparts of the maize kernel.

The color of the kernels is quite variable; yellow to orange is the mosttypical. However, white or red-brown varieties are also known. The hullconstitutes 5 to 6 percent of the kernel and consists mainly of cellulose andother insoluble polysaccharides. The proportion of germ is the highestamong the cereal grains—about 10 percent of the kernel mass. Most of thelipids and minerals are present in the germ. The protein content of the em-bryo is also high. There are two major types of starchy endosperm for corn,either horny (hard, translucent) or floury (soft, opaque). The horny endo-sperm is tightly compact, with few or no air spaces. Its starch granules, po-lygonal in shape, are held together by a matrix protein. In the opaque endo-sperm, the starch granules are spherical and are covered with a proteinmatrix, and there are many air spaces between the starch granules. Flintcorn varieties contain more horny than floury endosperm.

TABLE 11.5. Component parts of mature corn kernels and their chemicalcomposition

Part of kernelDry weight of whole

kernel (%)

Composition of kernel parts(% dry basis)

Starch Fat Protein Ash SugarWhole kernel 100.0 72.4 4.7 9.6 1.4 1.9Germ 11.5 8.3 34.4 18.5 10.3 11.0Endosperm 82.3 86.6 0.86 8.6 0.3 0.6Tip cup 0.8 5.3 3.8 9.7 1.7 1.5Pericarp 5.3 7.3 1.0 3.5 0.7 0.3

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The amylose content in normal maize starch ranges from 25 to 30 per-cent but can vary among cultivars and especially in corns with mutant genesamong the starch biosynthetic enzymes. The amylose content of starchfrom “high-amylose” (amylomaize) varieties can even reach up to 80 per-cent. In contrast, almost all of the starch derived from waxy corn, the mutantfor the wx waxy gene, is amylopectin. A third mutant, called sugary corn,contains significantly more highly branched amylopectins than normalcorn.

The protein content of the corn grain varies widely according to the vari-ety, agronomical conditions, and other environmental factors. It rangesfrom 6 to 18 percent (Lasztity, 1999). The Osborne procedure has beenwidely used for the fractional extraction of the proteins, resulting in albu-mins (~4 percent), globulins (~8 percent), prolamins (called “zeins”) (~50percent), and the polymeric glutelin fraction (~40 percent). The amino acidcomposition of the total corn-protein fraction is characterized by low con-tents of lysine and tryptophan. Genotypes with high levels of lysine(opaque-2 maize) are also known (Mertz, Nelson, and Bate, 1964), but theiracceptability has been hindered by their susceptibility to certain pests dueparticularly to their significantly higher moisture content. The nutritionaladvantages of some of the starch variants of maize endosperm have foundnew applications in various processed foods, such as are described byBranlard, Autran, and Monneveux (1989).

Genes coding the very polymorphic zein proteins are located on threedifferent chromosomes. The genes of the Z19 polypeptides (containing 210to 220 amino acid residues) are present in the region of about 30 crossoverunits on the short arms of chromosomes 7 and 9, while genes coding theZ22 polypeptides (with 240 to 245 amino acids) are scattered on both armsof chromosome 4. All of these polypeptides contain a 35 to 36 amino-acid-long N-terminal and a 10 amino-acid-long C-terminal region. The middleof the polypeptides consists of a repetitive region, built up of a 20-residue-long motif (Tatham, Shewry, and Belton, 1990).

Glutelin is a macromolecule of protein made up of diverse polypeptides(subunits) linked together via disulfide bonds. Based on solubility, the sub-units are grouped into three classes: (1) The water- (and alcohol-) solublesubunits (ASG proteins) are proline-rich prolamin-like polypeptides, alsocalled “gamma-zeins.” (2) A second group of polypeptides (C- and D-zeins) are soluble in alcohols but insoluble in water. (3) Polypeptides of thethird group are soluble in alkaline solutions. The most characterizedpolypeptide among the glutelin subunits is the ASG-1 protein. It consists ofabout 200 amino acids with a high proportion (8 percent) of cysteine resi-dues. The 11 amino-acid-residue-long N-terminal region is followed by arepetitive domain consisting of a highly conserved hexapeptide motive.

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The total lipid content of commercial corn hybrids averages 4 to 5 per-cent. Several hybrids, especially developed for oil production, can containas much as 12 to 20 percent lipids. Among numerous lipid components,corn oil’s major component (the triglycerides) has high nutritional value be-cause of its balanced fatty acid composition. It consists of around 12 per-cent palmitic acid, less than 4 percent stearic, 30 percent oleic, 40 percentlinoleic, and 3 percent linolenic acid.

Sorghum and Millets

Sorghum and a range of millet species are often grouped together be-cause of their similar niche of providing energy and protein to many peopledependent on growing them in dry tropical regions of Africa and Asia(Rooney, Kirleis, and Murty, 1987; Rooney, 1996). Sorghum is also knownas grain sorghum, great millet, milo, kaffir corn, and Guinea corn. Althoughsmall seeded, the millets have a hardiness that permits their cultivation as asubsistence crop under semiarid conditions, the most widely grown typesbeing pearl and finger millets (Table 11.1). Foods made from sorghum andmillets are various forms of bread (fermented and nonfermented), porridge,steamed foods, and various beverages.

Sorghum is mainly grown in the Western Hemisphere as a feed or indus-trial grain, but appropriate processing is necessary. This involves decorti-cation—removal of the outer layers that are mainly fiber. Thereafter, flouror grits are produced by milling; this may involve the use of stone mills inIndian villages, or hammer mills or roller milling in an industrial situation.Wet milling is preferred in many industrial settings, in a manner similar tothe wet milling of maize, often in preparation for further processing intostarch and glucose. The starch of sorghum is similar to that of maize, exceptthat it generally has a gelatinization point a few degrees higher than that ofmaize. Sorghum and the millets have (apparent) amylose contents of be-tween 20 and 30 percent. In addition, it has been possible to produce waxysorghum lines with virtually all the starch as amylopectin (Rooney, Kirleis,and Murty, 1987).

The protein content of sorghum grain is of the order of 10 percent, beingmainly prolamins (“kafirins”), but also glutelins (Figure 11.7) (Leite et al.,1999). Like many cereals, sorghum is deficient in lysine, threonine, andtryptophan, but high-lysine sorghum lines have been produced. The germ ofsorghum (about 15 percent of grain weight) contributes lipids to its energyvalue and also proteins of higher nutritional value. A unique feature of sor-ghum is that it produces tannins—polymeric polyphenols—located in thepericarp and testa layers of the seed coat. The tannins provide protection

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against insects and birds, and against weather damage by rain at harvest, butthese advantages are accompanied by losses in nutritional value. The vari-ous cultivars differ considerably in the extent of tannin production.

WHEAT-GRAIN QUALITY TRAITS: A MOLECULAR BASIS

The gluten proteins of wheat flour are the most studied of all the bio-chemical components of cereal grains. Cereal chemists have long been in-trigued by the unique properties of gluten, which was one of the first pro-teins to be prepared in reasonably pure form. This achievement is attributedto the Italian chemist Beccari, who demonstrated, early in the eighteenthcentury, that gluten could be washed from dough in a stream of running wa-ter (Bailey, 1941). This demonstration predated the invention of the name“protein” by a century. Much later again, the American chemist ThomasBurr Osborne shattered the concept that gluten is a single pure protein bydistinguishing between four proteins in wheat flour: albumin, globulin,gliadin, and glutenin. He demonstrated that the latter two are the compo-nents of the gluten fraction—distinct protein entities, differing in solubilityand in functional properties (Osborne and Vorhees, 1893)—thereby bring-ing the focus for dough quality onto the gluten proteins.

Dough Quality

A major quality attribute of wheat is its protein content. This single-number aspect of quality is used in determining grade quality and marketvalue. The importance for protein content stems from its reputation for be-ing an indicator of gluten content and dough quality. Both protein contentand dough properties are listed in Table 11.2 as being two of three basic fac-tors that determine the suitability of a grain sample for processing into spe-cific products. However, protein content is not synonymous with doughquality, because protein quality is distinct from protein content.

When wheat grain or flour is analyzed for protein content, the material iscompletely digested to ammonia (in the Kjeldahl method) or to nitrogen (byDumas analysis). As a result, there is complete destruction of informationabout protein structure and function; yet this is the approach most fre-quently used in practice to provide information about wheat protein. In rou-tine practice today, the determination of protein content may not actually in-volve digestion, but rather a correlative procedure such as near-infraredspectroscopy. Nevertheless, all the information about the vital grain proteinis often assumed by grain traders to be summarized in a single number—thepercentage of protein in the grain.

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The Levels of Integration at Which Gluten ProteinsMay Be Studied

In fact, protein content is the last of a series of levels of informationabout dough quality, as is shown in Figure 11.8. Each successive level ofstudy can be regarded as a window through which we peer to find out a partof the information about dough quality. All these views must be integratedinto a whole to obtain a full picture, which even then may not be complete.Unfortunately, to investigate gluten we must disrupt its structure, therebydestroying some of the information that is sought. For example, when acidhydrolysis is used to study amino acid composition, an enormous amount ofinformation is lost, though the results are nevertheless valuable for nutri-tional studies (Figure 11.6). Likewise, information about gluten structure islost when alkaline digestion is used to release ammonia from the amidegroups, but the result provides a quick guide to total protein content(Ronalds, 1974).

Ideally, the study of gluten-protein structure and function should start byexamination of the storage protein of the intact grain, before any aspects ofits structure have been damaged, even by milling, which is obviously an es-

Gli alleles

Low-S:High-S

Gliadin proteins

Glu alleles

HMW:LMWGlutenin subunits

Glutenin polymer size

Gliadin:Glutenin ratio

Gluten content

Amino acid composition

Amide content

Total nitrogen content

FIGURE 11.8. The degrees of formation and disruption of gluten, illustrating thelevels at which studies may be conducted on gluten-protein structure andfunction

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sential first step in most studies. Whole-grain studies may be possible usinga limited number of techniques, but it is difficult. For example, near-infra-red spectroscopy is being used to estimate not only protein content but alsoother factors that may indicate further information about processing quality.

An ideal approach is the microscopic study of the grain after cutting sec-tions or after breaking the grain in half for microscopic examination of mor-phological ultrastructure. This approach tells where the protein is locatedwithin the endosperm cells, its disproportionate distribution between theouter and inner layers of the endosperm cells, and possibly even informa-tion about where the different types of protein are laid down. Wheat-grainmorphology has been reviewed by Simmonds and O’Brien (1981) and byHeneen and Brismar (1987). Figure 11.9 shows the ultrastructure of parts ofthe wheat grain. It should be compared to the diagram of grain structure inFigure 11.5. The outer layers of bran can be seen covering the layer ofaleurone cells, with the endosperm cells on the inside, the endosperm beingthe material released by milling to produce white flour. The storage proteinof the endosperm is the source of gluten in dough, produced by the wettingand mixing action of dough formation. However, it is difficult for micro-scopic examination of the intact grain to provide major information aboutwhat aspects of gluten structure account for its importance in the provisionof dough properties.

FIGURE 11.9. Light micrograph of mature wheat grain, showing the outer layersof bran and the aleurone cells surrounding the starchy endosperm (Source: Pro-vided by W. P. Campbell, CSIRO, North Ryde, Australia.)

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The Polypeptides of Gluten

When gluten is solubilized, even by the gentlest methods, disruption ofmany of the bonds that account for its cohesion occurs, particularly thenoncovalent bonds such as hydrogen bonds, hydrophobic bonds, and vander Waals bonds. These many interactions have been reviewed in a book ed-ited by Hamer and Hoseney (1997). Rupture of the disulfide bonds of glutenproteins releases the individual polypeptides, and their composition be-comes accessible to study (by SDS gel electrophoresis or RP-HPLC) but welose the information about which parts of the polypeptides are linked to-gether by disulfide bonds. We also lose vital information about the sizes ofthe enormous polymers of the glutenin proteins. Figures 11.8 and 11.10show that the resulting polypeptides may come from either gliadin or glu-tenin fractions. The disulfide bonds of the gliadin proteins are mainlyintrachain, so that their rupture does not change their chain length. In con-trast to the gliadin fraction, many of the disulfide bonds of the glutenin frac-tion are between the individual chains, holding them into very large poly-mer structures. The contrast between these two types of components isshown in the diagrammatic representation of the molecules that are presentin dough (Figure 11.11).

RFLPPCR

SDS-PAGERP-HPLC

SE-HPLC

Multistackingelectrophoresis

FFF

Genes

Polypeptides

Gluteninpolymers

Very largepolymers

Glu1 Glu3 Gli1 Gli2

1A1B1D

6A6B6DHMW

10%LMW30%

Gliadins40%

FIGURE 11.10. Diagram of the major components of the gluten complex, includ-ing locations of genes and methods of analysis (RFLP = restriction-length frag-ment polymorphism; PCR = polymerase chain reaction; SDS-PAGE = sodiumdodecyl sulfate polyacrylamide gel electrophoresis; RP-HPLC = reversed phasehigh-performance liquid chromatography; SE-HPLC = size-exclusion high-per-formance liquid chromatography; FFF = field-flow fractionation)

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The Gliadin Proteins

Nevertheless, determination of the composition and proportions of thepolypeptides of gluten provides considerable information about the qualitypotential of the grain. The composition of the gliadin fraction has been usedfor decades as a means of identifying varieties (Wrigley, Autran, andBushuk, 1982), thereby providing vital information about the type of pro-cessing quality that has been “built in” by the breeder. The electrophoreticpatterns for gliadin proteins from a range of wheat varieties from variouscountries are compared in Figure 11.12. Gel electrophoresis of gliadin pro-teins has even been used to investigate the origins of archeological grainspecimens (Zeven, Doekes, and Kislev, 1975). Wheat grains likely to dateback to 500 B.C. were not suitable for analysis in this way, but useful resultscould be obtained for grain of about 175 years old.

As Figure 11.10 indicates, reversed-phase high performance liquid chro-matography (RP-HPLC) is also used to fractionate the polypeptides of thegluten proteins and thus to provide information about genotype and variety.The gliadin proteins are coded by the families of Gli-1 genes on the shortarms of the group-1 chromosomes and by the Gli-2 genes on the short armsof the group-6 chromosomes. The chromosomal locations of the genes for

LipidStarch granule

Lipid-binding protein

Glutenin

Gliadin

FIGURE 11.11. This diagrammatic representation of wheat-flour dough illus-trates gluten as a combination of the smaller monomeric gliadin molecules con-trasted with the very large polymeric structure of glutenin. (Source: Adapted fromWrigley, 1996.)

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the respective gluten proteins have been used as a major basis for develop-ing gluten-protein nomenclature (Wrigley, Bushuk, and Gupta, 1996). Thesubfractions of the gliadin class of monomeric gluten proteins have beendistinguished according to their mobilities in acidic gel electrophoresis.

Sulfur Deficiency and the Omega-Gliadin Proteins

The slowest-moving proteins, the omega-gliadins, are distinguishedfrom the alpha-, beta-, and gamma-gliadins by being almost completelylacking in the sulfur-containing amino acids cysteine and methionine (seeFigure 11.8). This difference is especially evident when analyzing the gli-adin proteins from grain grown under sulfur-deficient conditions (Randalland Wrigley, 1986), as is shown in Figure 11.13, using either single-dimen-sion acidic gel electrophoresis or a two-dimensional combination of elec-

FIGURE 11.12. Gliadin-protein patterns for a series of varieties of hexaploid(bread) wheat, showing how cathodic gel electrophoresis at pH 3 can be used todistinguish between varieties. Gliadin proteins were extracted from crushedgrains with 6 percent urea solution and the clarified extracts were applied to apolyacrylamide gel, whose concentration varied from 3 percent at the top to 13percent acrylamide at the bottom (negative electrode).From left to right, the vari-eties are Scout 66, Inia 66R, Capitole, Diplomat, Marquis, Chinese Spring,Eagle (Australian version), Halberd, Millewa, Olympic, Jabiru, and Lance.(Source: From C. W. Wrigley, J.-C. Autran, and W. Bushuk, 1982, Identification ofcereal varieties by gel electrophoresis of the grain proteins. Advances in CerealScience and Technology 5: 211-259. Reproduced by permission.)

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trophoretic methods. Moss and colleagues (1981) showed that dough qual-ity was severely impaired for grain from sulfur-deficient soil, especiallywith increased application of nitrogen fertilizer, due to changes in grain-protein composition resulting in the reduced synthesis of sulfur-rich pro-teins (Kettlewell et al., 1998; Wrigley et al., 1984). These differences relatenot only to the gliadin fraction, but also to the subfractions of glutenin(MacRitchie and Gupta, 1993). For both types of analysis of the gliadins(parts a and b of Figure 11.13), sulfur deficiency has affected the propor-tions of the components of slowest mobility (the omega-gliadins). Theseare more prominent on the left of the two-dimensional patterns (for sulfur-deficient grain). The four patterns on the left of Figure 11.13a are fromgrain grown with severe sulfur deficiency with adequate nitrogen fertilizer;the omega-gliadin bands are much more prominent than for the other sam-ples. These grain samples had low levels of sulfur (0.075, 0.081, 0.085, and0.091 percent S, left to right). The central group of patterns in Figure 11.13aare for grain fertilized with sulfur but not nitrogen (0.107, 0.111, 0.116, and0.122 percent S). The right-hand group of patterns are for “normal” grainwith adequate nitrogen and sulfur nutrition (0.135, 0.146, 0.157, and 0.161percent S). Studies of the sulfur-poor storage proteins of cereals have beenextended to rye and barley (Tatham and Shewry, 1995).

(a) (b)

FIGURE 11.13. The gliadin proteins of hexaploid (bread) wheat, illustrating theeffects of sulfur deficiency, indicated (a) by one-dimensional cathodic gel elec-trophoresis at pH 3, and (b) by two-dimensional isoelectric focusing followed bypH 3 gel electrophoresis. In part (a), the four lanes on the left correspond to graingrown with severe sulfur deficiency and adequate nitrogen fertilizer, the centralgroup corresponds to grains fertilized with sulfur but not nitrogen, and the fourlanes on the right are for grain with adequate nitrogen and sulfur nutrition. Thepattern on the left of part (b) shows the effect of sulfur deficiency, compared tothe control on the right.

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Glutenin Subunits

Gel electrophoresis of flour proteins in the presence of sodium dodecylsulfate (SDS) shows the glutenin proteins as an unresolved streak of proteinstaining (Wrigley, Gupta, and Bekes, 1993). This is understood to indicatethat the disulfide-linked polymers of glutenin subunits cover a wide rangeof molecular dimensions and that there is no ordered grouping of polymerdimensions. The sizes of these glutenin polymers range up into the tens ofmillions of Daltons, as has been shown by flow field-flow fractionation(FFF in Figure 11.9) (Stevenson and Preston, 1996; Wrigley, 1996).

However, when a reducing agent is added in the process of extractingflour (generally in the presence of SDS), disulfide bonds are broken andSDS-gel electrophoresis demonstrates that there are many discrete poly-peptide components, covering the molecular weight range from about30,000 to 120,000 Daltons (Figure 11.14). The larger polypeptides (the

FIGURE 11.14. SDS-gel electrophoresis of reduced glutenin polypeptides froma series of durum-wheat genotypes and near-isogenic lines (NIL) in which pairsof HMW subunits (numbered) have been incorporated. (1) Svevo-derived NILwith 5+10; (2) Svevo; (3) Lira-derived NIL with 5+10; (4) Lira; (5) Zenit-derivedNIL with 2+12; (6) Zenit; (7) Zenit-derived NIL with 5+10; (8) Zenit. (Source: FromD. Lafiandra et al., 1999, The formation of glutenin polymer in practice, CerealFoods World 44: 572-578. Reproduced with permission.)

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high-molecular weight [HMW] subunits) stand out above all the other flourcomponents; they have been allocated numbers according to their mobili-ties in the gel pattern, as is indicated in Figure 11.14. The list of numberedsubunits in Table 11.6 also indicates the corresponding allele designation(as lowercase italic letters). The HMW subunits are synthesized under thecontrol of Glu-1 genes on the long arms of wheat chromosomes 1A, 1B, and1D, as indicated in Figure 11.10. The HMW subunits often appear as pairs(e.g., 5 with 10, 2 with 12, and 17 with 18), because their synthesis is con-trolled by corresponding pairs of genes at the Glu-1 locus. This is also indi-cated in Table 11.6, which lists the correspondence between the subunitnumbers and the gene designations (alleles). For example, subunit 1 is syn-thesized under the control of allele a (also designated as Glu-A1a), and sub-units 5 and 10, under the control of the allele Glu-D1d.

The subunits of glutenin have been reported to have a central domainwith a highly stable spiral structure, stabilized by extensive inter-turn hy-drogen bonding, involving the glutamine side chains of the repeating aminoacid sequences which are rich in glutamine, proline, and glycine (Kasarda,King, and Kumosinski, 1994). This spiral structure is proposed as a meansby which the glutenin subunits may confer elastic properties in dough; it isthe reason that spiral structures have been used in Figure 11.11 to depict themain central region of the glutenin subunits.

TABLE 11.6.Glu-1 dough-quality scores assigned to HMW-glutenin subunits andcorresponding alleles

Glu-1 Glu-A1 Glu-B1 Glu-D1

Score Allele Subunit Allele Subunit Allele Subunit

4 d 5+103 a 1 i 17+183 b 2* b 7+83 f 13+162 a 2+122 b 3+121 c Null a 7 c 4+121 d 6+81 e 20

Source: According to Payne, 1987.Note: A high score (maximum of 10) indicates the prediction of strong doughproperties.

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The low-molecular weight (LMW) subunits, which outweigh the HMWsubunits by three to one, appear farther down the SDS-gel electrophoreticpattern, but their presence is normally disguised by the overlapping bandsof non-glutenin components, mainly gliadins. This difficulty has been over-come by a two-step method of electrophoresis or by selective extractionprocedures (Gupta and Shepherd, 1990). The LMW subunits are coded bythe families of Glu-3 genes on the short arms of the group-1 chromosomes,tightly linked to the genes for some of the gliadin proteins (the Gli-1 alleles,shown in Figure 11.10). Accordingly, a unified classification system hasbeen proposed for the alleles of the gliadins (Gli-1) and LMW-subunits ofglutenin (Glu-3) (Jackson et al., 1997).

Prediction of Dough Quality from Glutenin Allelic Constitution

The functional properties of the HMW subunits have been deduced by arange of methods. Initial indications came from quality analyses of progenyfrom crosses between wheats that differed in glutenin composition (Payne,Corfield, and Blackman, 1979). In addition, correlations for subunit com-position to quality have been studied for many sets of wheat genotypes(e.g., Gupta, Bekes, and Wrigley, 1991; Gupta et al., 1994), and from durumlines into which were incorporated pairs of glutenin polypeptides (Lafian-dra et al., 1999). A more direct approach has been the analysis of genotypesdevised to have some or all of the HMW subunits absent (Lawrence,MacRitchie, and Wrigley, 1988), as is shown in Figure 11.15. In this case,the loss of any one of the subunits reduced dough quality, but of the threesets of glutenin subunits, the loss of the 5+10 pair (for lines correspondingto lanes b, c, and d in Figure 11.15) caused the most dramatic loss of doughproperties. Loss of two or more subunits caused progressively further lossesof quality. Dough-forming ability was lost completely with the loss of allHMW subunits, even though the full complement of LMW subunits was re-tained. Other approaches have involved the development of lines havingspecific interchanges of subunits and most recently by the isolation of indi-vidual subunits (or their genes) for direct testing by incorporation into theglutenin structure of a common parent dough (Bekes et al., 1994; Barroet al., 1997). In addition, confirmatory data about the contributions todough quality of these proteins has been obtained by transformation stud-ies, in which the genes for the polypeptides have been inserted into a com-mon wheat background (e.g., one of the lines that lack the full complementof subunits in Figure 11.15) (Barro et al., 1997).

The contributions of the individual HMW subunits of glutenin have beenranked according to their additive effects by Payne (1987). These are set out

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systematically in Table 11.6 as score numbers. The total score for a varietyis based on the set of its three HMW subunits as the sum of the contributionfrom each subunit (one for each of the three genomes of wheat—A, B, andD). For example, a score of 10 (highest possible) would be obtained for al-leles a, i, and d (in each of the A, B, and D genomes, corresponding to pro-tein subunits 1, 17+18, and 5+10), derived from individual scores of 3, 3,and 4, respectively, according to Table 11.6. As these rankings indicate theimportance of the subunits in relation to dough properties, they can be usedin selecting breeding lines for processing quality. This strategy of predict-ing dough quality is valuable because the Glu-1 constitution of manywheats is now known. Catalogs of these subunits have been reported formany national collections of wheats (e.g., for Canada by Lukow, Payne, andTkachuk, 1989; for Australia by Lawrence, 1986), and the worldwide dis-tribution of these alleles has been surveyed by Morgunov and colleagues(1993).

FIGURE 11.15. SDS-gel electrophoresis of reduced glutenin polypeptides froma series of wheat genotypes and near-isogenic lines (NIL) in which specific com-binations of HMW subunits have been removed (Source: From G. J. Lawrence,F. MacRitchie, and C. W. Wrigley, 1988, Dough and baking quality of wheat linesdeficient for glutenin subunits controlled by Glu-A1, Glu-B1, and Glu-D1 loci.Journal of Cereal Science 7: 109-112. Reproduced with permission from Aca-demic Press.)

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However, in several of these surveys of national wheats, the Glu-1 qual-ity score accounts for only part of the variation in dough or baking quality,e.g., for only about 60 percent of the variation in bread-making quality for67 Canadian varieties (Lukow, Payne, and Tkachuk, 1989) and more or lessfor other national collections (MacRitchie, du Cros, and Wrigley, 1990).This is partly because the HMW-subunits of glutenin constitute only aboutone-quarter of the total glutenin protein; the remaining portion is made upof the LMW subunits. Nevertheless, the HMW subunits appear to have adisproportionately larger contribution to dough properties, probably be-cause of their larger size. The LMW subunits of glutenin also confer a mod-erating effect on dough properties, but this contribution has been more diffi-cult to quantify, due to their greater diversity and the relative difficulty inanalyzing for LMW-subunit composition. Gupta and colleauges (1994) an-alyzed the HMW and LMW subunits of the glutenin proteins of 74 recom-binant inbred lines that were homozygous for these alleles. They found thatabout 80 percent of the variability in dough strength (measured as Rmax inthe Extensigraph) could be accounted for if both LMW and HMW subunitcomposition were considered, but that predictability was much worse ifonly one or other aspect of glutenin subunit composition were considered.Cornish, Panozzo, and Wrigley (1999) and Cornish, Griffin, and Wrigley(2000) have shown how breeding for specific quality traits can be manipu-lated by consideration of overall glutenin-subunit composition.

The pasta-cooking quality of the durum wheats might be expected to bereflected in the composition of their HMW glutenin subunits. However,most studies have indicated that the LMW subunits of durum glutenin havebetter predictive value than their HMW subunits (Branlard, Autran, andMonneveux, 1989; Feillet et al., 1989; Pogna et al., 1988).

The Size Distribution of Glutenin Polymers

The most recent studies aimed at relating gluten composition to func-tional properties in dough have concentrated on the size distribution of thenative polymers of glutenin. Indeed, this concept may be extended to a con-sideration of size distribution for the full complement of gluten proteins,thereby including the monomeric gliadin proteins and the ratio of gliadin toglutenin proteins. Size distribution for the gluten proteins thus extends fromthe relatively small gliadin proteins up into the tens of millions of Daltonsfor the largest of the glutenin polymers (Southan and MacRitchie, 1999;Zhu and Khan, 2001). It appears to be these very large molecules that con-tribute the resistance to extension which is critical to dough strength, whilethe range of smaller proteins provides the balance of dough viscosity. Fur-

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thermore, some of the subunits of glutenin seem to be more effective thanothers in contributing to the functional properties of glutenin, possibly byproviding extra size (length) to its polymeric structure. A reduction in sizedistribution of glutenin has been shown to be a critical factor in explainingthe loss of dough strength (Lafiandra et al., 1999) that is often associatedwith heat stress during grain filling in the field (Blumenthal, Barlow, andWrigley, 1993; Ciaffi et al., 1996; Corbellini et al., 1997, 1998).

GRAIN HARDNESS

The need for appropriate dough properties is complemented by that ofgrain hardness (Table 11.2). In the technological sequence of grain utiliza-tion, grain hardness has its first major role in grain milling. When softwheats are milled, the endosperm falls apart readily, allowing the individualstarch granules to separate from one another, with minimal damage to thesurface of the granules. As a result, the remnants of the membranes sur-rounding the granules remain undamaged, providing a degree of resistanceto the attack of starch-degrading enzymes. On the other hand, when hardwheats are milled, the endosperm tends to hold together, and fractures mayoccur right through the starch granules. As a result, hydrolytic enzymeshave ready access to the starch polymers inside the granules during the laterstages of processing, such as fermentation and baking. Furthermore, waterabsorption is likely to be higher due to the damage to the granules.

Grain hardness is largely determined by one major gene, Ha, located onthe short arm of chromosome 5D (Turner et al., 1999). One of the proteinsassociated with the starch granules (“friabilin”) has been implicated as be-ing a potential marker of grain hardness (Morrison et al., 1992). This pro-tein (of about 15,000 Daltons) is present on the surface of starch granuleswashed from soft wheats and is absent or present in smaller amounts on thesurface of granules from hard wheats. It is a member of a family of hydro-lase inhibitors and puroindolines (Rahman et al., 1994), particularly puro-indolines a and b (Giroux and Morris, 1997, 1998). More recent results in-dicate that biochemical factors in addition to the puroindolines are involvedin grain hardness, but the presence of puroindoline b has been found to cor-relate with bread-making quality (as loaf volume) (Igrejas et al., 2001).

STARCH PROPERTIES

Other specific proteins of the starch granule have been associated withvariations in the functional properties of wheat starch, particularly the ratio

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of amylose to amylopectin (Rahman et al., 2000). The extent of synthesis ofthe smaller linear molecule, amylose, has been shown to be determined bythree isoforms of the enzyme granule-bound starch synthase (GBSS) (Naka-mura et al., 1993). These isoforms are coded by homologous genes on chro-mosomes 7A, 7D, and 4A, designated as the “waxy” genes Wx-A1, Wx-D1,and Wx-B1, respectively. A wheat genotype that lacks all three of thesegenes produces no amylose due to the absence of the GBSS enzymes; all itsstarch is of the amylopectin type—highly branched starch with large mo-lecular weight. This is known as waxy wheat because of the translucent ap-pearance of the endosperm (Zhao, Shariflou, et al., 1998). The productionof the waxy genotype is new in wheat, whereas waxy types have long beenknown in rice, maize, barley, and sorghum. The starch from waxy wheat hasdistinct functional properties, namely, greater hot-paste viscosity whenheated with water and better freeze-thaw stability for the resulting gel,which is optically clear.

The other extreme genotype, having all three isoforms of GBSS, is themost common in wheat; it produces 20 to 25 percent amylose, the remain-der being amylopectin. All intermediate combinations of the three geneshave been produced, lacking one or two of the genes; these contain interme-diate levels of amylose (Yamamori and Quynh, 2000; Zhao, Shariflou,et al., 1998). The genotype lacking the 4A gene (Wx-B1) produces wheatshaving starch properties that are particularly suited to the production of var-ious types of noodles (Zhao, Batey, et al., 1998). Null-4A (Wx-B1) geno-types have starch properties with higher viscosity (when heated in water)and greater swelling power. These properties are conducive to the produc-tion of many types of noodles, especially the Japanese udon type. The storyof the development of the range of reduced-amylose wheats has been re-viewed by Seib (2000).

A recent breakthrough in starch properties has been the ability to manip-ulate the proportions of the A-type (large) starch granules versus the smallB granules (Stoddard, 1999). Earlier studies had been hampered by an ap-parent lack of genetic diversity with respect to this trait. In addition, it hadbeen assumed to be of relatively little importance for baking quality, al-though granule-size distribution has been reported to contribute to doughproperties and to water binding (Rasper and deMan, 1980). However, granule-size distribution is critical to the starch-gluten industry; a high proportion ofA granules means a better yield of high-quality starch, and the consequentlower proportion of the small B granules means less starch appearing in ef-fluent streams, thereby incurring reduced disposal problems (Rahman et al.,2000). It now appears likely that wheat cultivars can be developed with awider range of size distribution in their starch granules (Stoddard, 1999).

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CONCLUSION

Humankind is critically dependent on the cereal grains for nutrition—today as much as in the early days of civilization. Today, however, we havethe great advantage of genotypes that are well adapted to the sites of cultiva-tion, yielding grain suited to the specific requirements of processing andconsumption. These advantages have been won over millennia of effort. Inprehistory and in more recent times, this involved the careful selection ofplants whose grain appeared to be better for grinding and baking. Duringthe past century, the range of genetic diversity has been extended by cross-breeding, permitting desirable traits to be combined from a few parents intoone genotype. These methods, now considered to be conventional, maynext be extended by genetic-engineering technology, with even wider ge-netic diversity being possible from which to make selections for improve-ment. Nevertheless, even the imaginative use of the more conventionalmethods is still proving effective in producing improved genotypes, as indi-cated by the examples described for better dough and starch properties inwheat.

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Kasarda, D.D., King, G., and Kumosinski, T.F. (1994). Comparison of spiral struc-tures in wheat high molecular weight glutenin subunits and elastin by molecularmodeling. In Kumosinski, T.F. and Lieberman, M.N. (Eds.), ACS SymposiumSeries No. 576. Molecular Modelling: From Virtual Tools to Real Problems(pp. 209-220).Washington, DC: American Chemical Society.

Kettlewell, P.S., Griffiths, M.W., Hocking, T.J., and Wallington, D.J. (1998). De-pendence of wheat dough extensibility on flour sulphur and nitrogen concentra-tions and the influence of foliar-applied sulphur and nitrogen fertilisers. Journalof Cereal Science 28: 15-23.

Krishnan, P.G., Reeves, D.L., Kephart, K.D., Thiex, N., and Calimente, M. (2000).Robustness of near infrared reflectance spectroscopy measurement of fatty acidsand oil concentrations in oats. Cereal Foods World 45: 513-519.

Lafiandra, D., Masci, S., Blumenthal, C., and Wrigley, C.W. (1999). The formationof glutenin polymer in practice. Cereal Foods World 44: 572-578.

Lasztity, R. (1999). Cereal Chemistry. Budapest, Hungary: Akadémiai Könyv-kiadó.

Lawrence, G.J. (1986). The high molecular weight subunit composition of Austra-lian wheat cultivars. Australian Journal of Agricultural Research 37: 125-133.

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Lawrence, G.J., MacRitchie, F., and Wrigley, C.W. (1988). Dough and baking qual-ity of wheat lines deficient for glutenin subunits controlled by Glu-A1, Glu-B1and Glu-D1 loci. Journal of Cereal Science 7: 109-112.

Lehmann, J.W. (1996). Case history of grain amaranth as an alternative crop. CerealFoods World 41: 399-411.

Leite, A., Neto, G.C., Vettore, A.L., Yunes, J.A., and Arruda, P. (1999). Theprolamins of sorghum, Coix and millets. In Shewry, P.R. and Casey, R. (Eds.),Seed Proteins (pp. 141-157). Dordrecht, the Netherlands: Kluwer AcademicPublishers.

Lukow, O.M., Payne, P.I., and Tkachuk, R. (1989). The HMW glutenin subunitcomposition of Canadian wheat cultivars and their association with bread-mak-ing quality. Journal of the Science of Food and Agriculture 47: 451-460.

MacGregor, A.W. and Bhatty, R.S. (1993). Barley: Chemistry and Technology.St. Paul, MN: American Association of Cereal Chemists.

MacRitchie, F., du Cros, D.L., and Wrigley, C.W. (1990). Flour polypeptides re-lated to wheat quality. Advances in Cereal Science and Technology 10: 79-145.

MacRitchie, F. and Gupta, R.B. (1993). Functionality-composition relationships ofwheat flour as a result of variation in sulfur availability. Australian Journal ofAgricultural Research 44: 1767-1774.

Malkki, Y. (2001). Physical properties of dietary fiber as keys to physiologicalfunctions. Cereal Foods World 46: 196-199.

Martin, A., Alvarez, J.B., Martin, L.M., Barro, F., and Ballesteros, J. (1999). Thedevelopment of tritordeum: A novel cereal for food processing. Journal of Ce-real Science 30: 85-95.

Mertz, E., Nelson, O., and Bate, L.S. (1964). Mutant gene that changes compositionand increases lysine content of maize endosperm. Science 154: 279-281.

Morgunov, A.I., Pena, R.J., Crossa, J., and Rajaram, S. (1993). Worldwide distribu-tion of Glu-1 alleles in bread wheat. Journal of Genetics and Breeding 47: 53-60.

Morris, C.F. and Rose, S.P. (1996). Wheat. In Henry, R.J. and Kettlewell, P.S.(Eds.), Cereal Grain Quality (pp. 3-54). London: Chapman and Hall.

Morrison, W.R., Greenwell, P., Law, C.N., and Sulaiman, B.D. (1992). Occurrenceof friabilin, a low molecular weight protein associated with grain softness, onstarch granules isolated from some wheats and related species. Journal of CerealScience 15: 143-149.

Moss, H.J., Miskelly, D.M., and Moss, R. (1986). The effect of alkaline conditionson the properties of wheat flour dough and Cantonese-style noodles. Journal ofCereal Science 4: 261-268.

Moss, H.J., Wrigley, C.W., MacRitchie, F., and Randall, P.J. (1981). Sulfur and ni-trogen fertiliser effects: II. Influence on grain quality. Australian Journal of Ag-ricultural Research 32: 213-226.

Nakamura, T., Yamamori, M., Hirano, H., and Hidaka, S. (1993). Decrease in waxy(Wx) in two common wheat cultivars with low amylose content. Plant Breeding111: 99-105.

Nelson, A.L. (2001). Properties of high-fiber ingredients. Cereal Foods World 46:93-97.

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Osborne, T.B. and Vorhees, C.G. (1893). The proteids of the wheat kernel. Journalof the American Chemical Society 15: 392-471.

Payne, P.I. (1987). Genetics of wheat storage proteins and the effect of allelic varia-tion on bread-making quality. Annual Review of Plant Physiology 38: 141-153.

Payne, P.I., Corfield, K.G., and Blackman, J.A. (1979). Identification of a high-molecular-weight subunit of glutenin whose presence correlates with bread-making quality in wheats of related pedigree. Theoretical and Applied Genetics55: 153-159.

Pogna, N., Lafiandra, D., Feillet, P., and Autran, J.C. (1988). Evidence for a directcausal effect of low molecular weight subunits of glutenins on gluten visco-elasticity in durum wheats. Journal of Cereal Science 7: 211-214.

Qarooni, J. (1996). Wheat characteristics for flat breads. Cereal Foods World 41:391-395.

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Rahman, S., Jolly, C.J., Skerritt, J.H., and Walloscheck, A. (1994). Cloning of awheat 15-kDa grain softness protein (GSP): GSP is a mixture of puroindoline-like polypeptides. European Journal of Biochemistry 223: 917-925.

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Rasper, V.F. and deMan, J.M. (1980). Effect of granule size of substituted starcheson the rheological character of composite doughs. Cereal Chemistry 50: 331-340.

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Rooney, L., Kirleis, A.W., and Murty, D.S. (1987). Traditional foods for sorghum:Their production, evaluation, and nutritional value. Advances in Cereal Scienceand Technology 8: 317-353.

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Shotwell, M.A. (1999). Oat globulins. In P.R. and Casey, R. (Eds.), Seed Proteins(pp. 389-400). Dordrecht, the Netherlands: Kluwer Academic Publishers.

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Wrigley, C.W. (1994). Developing better strategies to improve grain quality forwheat. Australian Journal of Agricultural Research 45: 1-17

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Wrigley, C.W., Bushuk, W., and Gupta, R. (1996). Nomenclature: Establishing acommon gluten language. In Wrigley, C.W. (Ed.), Gluten ’96 (pp. 403-407).Melbourne: Royal Australian Chemical Institute.

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Yamamori, M. and Quynh, N.T. (2000). Differential effects of Wx-A1, -B1 and -D1protein deficiencies on apparent amylose content and starch pasting properties incommon wheat. Theoretical and Applied Genetics 100: 32-38.

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Zhao, X.C., Batey, I.L., Sharp, P.J., Crosbie, G., Barclay, I., Wilson, R., Morell,M.K., and Appels, R. (1998). A single locus associated with starch granule prop-erties and noodle quality in wheat. Journal of Cereal Science 27: 7-13.

Zhao, X.C., Shariflou, M.R., Good, G., and Sharp, P.J. (1998). Developing waxywheat cultivars: Wx null alleles and molecular markers. In Slinkar, A.E. (Ed.),Proceedings of the Ninth International Wheat Genetics Symposium, Volume 1(pp. 254-256). Saskatoon, Canada: University of Saskatchewan.

Zhu, J. and Khan, K. (2001). Effects of genotype and environment on glutenin poly-mers and breadmaking quality. Cereal Chemistry 78: 125-130.

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Chapter 12

Grain Quality in Oil CropsGrain Quality in Oil Crops

Leonardo VelascoBegoña Pérez-Vich

José M. Fernández-Martínez

INTRODUCTION

Oil crops are domesticated plants whose seeds or fruits are valued mainlyfor the oils or fats that are extracted from them. The difference between oilsand fats is merely the consistency at room temperature. We speak of an oil ifit is liquid at the prevailing temperature of the region where it is producedand of a fat if it is normally solid (Hatje, 1989). Oil crops include both an-nual (usually called oilseeds) and perennial plants from a wide range ofplant families. Table 12.1 lists the most important oil crops of the world aswell as their most relevant properties and uses.

About 8 percent of the world production of oil crops is directly con-sumed as food (e.g., groundnuts), and about 6 percent is used for seed andanimal feed. The remaining production is processed into oil (Food and Ag-riculture Organization of the United Nations [FAO], 2001). The oil/fat con-tent of oil crops varies widely, from about 10 percent of the weight in coco-nuts to over 50 percent in palm kernels. An important aspect of oil crops isthat they yield two products of economic value: the oil or fat and the oilmeal(also known as oilcake) that remains after oil extraction. Such oilmeals usu-ally contain a high crude protein content (from 20 percent in palm kernelmeal to almost 50 percent in soybean meal) and are mainly used as proteinsupplements for animal feed (Table 12.2). Oilseed meals are also used asfertilizers and soil improvers in many areas of the world (Bell, 1989). Inmost oilcrops the oil contributes a major percentage to the total value of theproducts. In soybean, however, the meal accounts for approximately 60 to70 percent of the value of the seed (Smith and Huyser, 1987).

Vegetable oils and fats have two main uses: human consumption andtechnical or industrial applications. Vegetable oils account for about 70 per-cent of the world edible fat production, the rest coming from animal fats.

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TABLE 12.1. Main oil crops of the world

Oil crop FamilyPlanthabit

Plantpart Oil/fat

Annualproductionof seed/fruit(× 1000 t)

Annualproductionof oil/fat(× 1000 t)

Soybean [Glycine max (L.)Merr.]

Fabaceae Annual Seed Oil 161,993 23,235

Palm (Elaeis guineensisJacq.)

Palmae Perennial Fruit Fat 116,315 21,951

Rapeseed (Brassica spp.) Brassicaceae Annual Seed Oil 40,193 12,362Sunflower (Helianthusannuus L.)

Asteraceae Annual Seed Oil 26,800 9,513

Groundnuta (Arachishypogaea L.)

Fabaceae Annual Seed Oil 34,507 4,557

Cottonseed (Gossypiumspp.)

Malvaceae Annual Seed Oil 54,143 3,849

Coconut (Cocos nuciferaL.)

Palmae Perennial Seed Oilb 46,482 3,319

Palm kernelc (Elaeisguineensis Jacq.)

Palmae Perennial Seed Fat 6,318 2,695

Olive (Olea europaea L.) Oleaceae Perennial Fruit Oil 13,599 2,457

Source: FAO, 2001.aIn shellbLiquid at the temperature of tropical areas, where it is produced, but solid in temperate regionscOil from palm fruits obtained both from the pulp or mesocarp (palm oil) and the kernel (palm kernel oil)

TABLE 12.2. Typical oil content of the seeds (percent dry seed weight), protein(N × 6.25), crude fiber (CF), and nitrogen-free extract (NFE) contents of thedefatted meals (dry meal weight), and lysine and methionine + cysteine concen-trations in the proteins (percent total protein) of the principal oilseeds

Oilseed Oil Protein* CF* NFE* Lysine Met+Cys

Canola 45 39 10 37 6.0 3.0

Cottonseed 16 42 13 35 4.1 3.3

Linseed 40 36 10 42 3.3 3.6

Peanut 50 47 14 28 3.8 2.7

Safflower 35 41 9 29 3.3 3.1

Soybean 18 49 6 34 6.2 2.0

Sunflower 45 47 12 27 3.0 2.8

Source: Data from Weiss, 1983; Bell, 1989; Vohra, 1989; Dorrell and Vick, 1997.*Data for dehulled sunflower and safflower meals and partly dehulled peanut meal

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Oils and fats are a vital component of the human diet because they are im-portant sources of energy, act as carriers for fat-soluble vitamins, and pro-vide the organism with essential fatty acids (Vles and Gottenbos, 1989).Human fat consumption has two main components: the so-called visible fat(butter, margarine, salad oil, cooking oil) and invisible fat (milk, meat,cheese, pastry, snacks, bread, nuts). Apart from food uses, large quantitiesof vegetable oils are directed to nonfood applications. They are used as mo-tor fuels (biodiesel) and lubricants as well as for many applications in theoleochemical industry (detergents, soaps, surfactants, emulsifiers, cosme-tics, etc.).

Breeding advances in the improvement of oil and meal properties of oilcrops have had great market impacts. Rapeseed is probably the most re-markable example. It traditionally contained a toxic fatty acid in the oil(erucic acid) as well as antinutritive compounds in the meal (glucosino-lates). In the 1970s, plant breeders were able to develop new types, laternamed canola, that essentially were free from both factors. Such improve-ment led to an enormous expansion of acreage and, consequently, to a con-siderable increase of rapeseed oil, meal, and products in the world market(Becker, Löptien, and Röbbelen, 1999). Similarly, the outstanding resultsof Russian breeders in raising the oil concentration of sunflower seeds wasone of the keys for the expansion of sunflower as one of the most importantoil crops in the world (Putt, 1997).

COMPONENTS OF GRAIN QUALITY IN OIL CROPSAND FACTORS INFLUENCING THEM

Oilseeds contain large amounts of food reserves which support the initialdevelopment of the seedling. Unlike cereals and legumes, in which the mainfood reserves are carbohydrates and proteins, respectively, most oilseedscontain oil as the main seed reserve (Table 12.2). The oil reserves are laiddown in discrete subcellular organelles, the oil bodies, concentrated in theembryonic tissues. In the case of castor bean, however, oil bodies are mainlylocated in the endosperm (Bewley and Black, 1978).

Grain quality of oil crops has three main components: the oil content ofthe grain, the quality of the oil, and the quality of the oilmeal that remainsafter oil extraction. The quality of the oil is mainly determined by its com-position in triacylglycerols and fatty acids as well as by the total content andcharacteristics of the antioxidant substances present in the oil. The qualityof the meal is largely defined by the fiber content, protein content, and itsnutritional value, as well as by the absence of toxic and antinutritional com-

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pounds. All these components will be described in detail in the correspond-ing sections of this chapter.

Grain quality of an oil plant is a combination of its genotypic constitu-tion and the expression of the genotype in a given environment. The latterdepends on not only environmental factors such as light and temperature,but also on intrinsic plant characteristics such as mode of reproduction(self-pollination versus outcrossing) and on the relative contribution of theparent genotypes to the trait (gametophytic versus sporophytic control). Forexample, oil and protein contents are mainly determined by the genotype ofthe grain-bearing plant (maternal or sporophytic control), whereas the fattyacid composition of seed oil is mainly determined by the genotype of thedeveloping embryo (embryogenic or gametophytic control).

There is some variation in the degree to which the components of grainquality are affected by genotypic and environmental factors. As a generalrule, grain quality traits can be divided into quantitative traits, if they arepolygenic and their expression is largely affected by the environment inwhich the plants are grown, and qualitative traits, if their expression is rela-tively independent from the environment and determined by major genes.Examples of quantitative traits are the oil and protein contents and the totalconcentration of antioxidant or antinutritional compounds. Examples ofqualitative traits are the oil fatty acid profile and the tocopherol profile.

In general, it can be stated that all factors affecting the general plant andgrain development also influence grain quality. The influence of tempera-ture (Canvin, 1965), light intensity (Dybing and Zimmerman, 1966), andenvironmental stress (Bouchereau et al., 1996; Velasco, Fernández-Martínez,and De Haro, 2001) on grain quality components is well documented. In ad-dition, intrinsic grain characteristics such as hull percentage, grain size, andgrain color can also affect grain quality. In sunflower, about two-thirds ofthe increase in achene oil content has resulted from a reduction in hull per-centage, and about one-third from an increase in kernel oil content (Fickand Miller, 1997). In rapeseed, both yellow-coated grains and larger grainscontain a greater proportion of meat to hull, which has been associated withgreater oil and protein contents and a lower crude fiber content, resulting ina better digestibility of the oilmeal (Bell and Shires, 1982; Jensen, Liu, andEggum, 1995).

OIL QUALITY

Basically, vegetable oils are made up of triacylglycerol molecules, whichusually constitute more than 95 percent of the oil weight. The triacylgly-cerol contains one glycerol and three fatty acid molecules. There are several

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types of fatty acids, mainly differing in the number of carbon atoms and/ornumber and position of double bonds in the carbon chain. Both the fattyacid profile of the oil and the pattern of distribution of fatty acids within thetriacylglycerol molecule constitute the principal factors determining thequality of vegetable oils, i.e., their physical, chemical, physiological, nutri-tional, and technological properties (Somerville, 1991; Padley, Gunstone,and Harwood, 1994).

Vegetable oils also contain a number of minor compounds, includinglipids (polar lipids, mono- and diacylglicerols, free fatty acids, etc.) andlipid-soluble compounds. The most relevant of the latter are a series of de-rivatives of isoprene, comprising sterols, tocopherols, carotenoids, andchlorophylls, some of which are of paramount importance for oil quality be-cause of their antioxidant properties.

Fatty Acid Composition of Oils

The most common classification of fatty acids is based on the number ofdouble bonds present in the molecule. Thus, fatty acids are classified intosaturated if they do not contain double bonds, monounsaturated if they con-tain one double bond, and polyunsaturated if they possess two or more dou-ble bonds in the molecule. Saturated fatty acids are major components oflipids that are solid at room temperature (fats), whereas unsaturated fattyacids are the major components of liquid lipids (oils). Fatty acids are repre-sented by a system of abbreviated nomenclature that designates chainlength and degree of unsaturation. For example 18:0 designates an 18-car-bon saturated fatty acid (stearic acid) whereas 18:3 indicates three doublebonds (Lobb, 1992). In addition, the abbreviated information for unsatu-rated fatty acids includes the (n-x) symbol, where x is the position of thefirst unsaturated carbon from the methyl end in the fatty acid molecule. Thisposition is of utmost importance for the nutritional and pharmaceuticalproperties of fatty acids (Åppelqvist, 1989). With this nomenclature, thesymbol 18:3 (n-3) refers to alpha-linolenic acid, whereas 18:3 (n-6) is usedfor gamma-linolenic acid. The main fatty acids in vegetable oils are listed inTable 12.3.

The formation of the major fatty acids in oilseeds starts by de novo syn-thesis of 16-carbon and 18-carbon fatty acids in the cell plastid (Somervilleand Browse, 1991) through the combined activity of different enzymes us-ing acetyl-CoA and malonyl-CoA as precursors (Figure 12.1). In addition,acyl carrier protein (ACP) is a required cofactor to which the intermediatemetabolites in the plastid pathway are attached as thioesters. The first stepin the pathway is the transfer of malonate from coenzyme A (CoA) to ACP.

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Three condensing enzymes then utilize malonyl-ACP as the 2-carbon donorfor elongation of the growing acyl chain. The first condensation reaction isbetween malonyl-ACP and acetyl-CoA by the action of 3-ketoacyl-ACPsynthase III (KAS III) (Jaworski, Clough, and Barnum, 1989). Subsequentcondensations are between malonyl-ACP and acyl-ACP intermediates andare catalyzed by KAS I. The final 2-carbon elongation occurring in plastids isfrom 16:0 to 18:0 and requires KAS II. After each condensation, the 3-keto-acyl-ACP intermediates are reduced, dehydrated, and reduced again toyield the saturated acyl-ACP intermediates (Harwood, 1996). This fattyacid synthase (FAS) system is similar to the type II fatty acid synthase ofEscherichia coli, as each of its component enzymes can be isolated sepa-rately. Finally, 18:0 is efficiently desaturated to 18:1 by the stearoyl-ACPdesaturase (SAD).

The 16:0-ACP, 18:0-ACP, and 18:1-ACP formed in the plastid are hydro-lized to free fatty acids by acyl-ACP thioesterases. The hydrolysis of theacyl-ACP thioester bond by the thioesterases implies the termination of theacyl chain elongation. Acyl-ACP thioesterases have been divided into FatAtype (with oleoyl-ACP as preferred substrate) and FatB type (with saturated

TABLE 12.3. Main fatty acids present in vegetable oils

Common name Systematic name SymbolCaprylic Octanoic 8:0Capric Decanoic 10:0Lauric Dodecanoic 12:0Myristic Tetradecanoic 14:0Palmitic Hexadecanoic 16:0Palmitoleic cis-9-Hexadecenoic 16:1 (n-7)Stearic Octadecanoic 18:0Oleic cis-9-Octadecenoic 18:1 (n-9)Ricinoleic 12D(R)-Hydroxy-9-Octadecenoic 18:1-OHLinoleic 9,12-Octadecadienoic 18:2 (n-6)Alpha-linolenic 9,12,15-Octadecatrienoic 18:3 (n-3)Gamma-linolenic 6,9,12-Octadecatrienoic 18:3 (n-6)Arachidic Eicosanoic 20:0Behenic Docosanoic 22:0Gadoleic cis-9-Eicosenoic 20:1 (n-11)Eicosenoic cis-11-Eicosenoic 20:1 (n-9)Erucic cis-13-Docosenoic 22:1 (n-9)

Source: Data from Lobb, 1992 and Åppelqvist, 1989.

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substrates preferred) (Jones, Davies, and Voelker, 1995). Thioesterases playan important role in determining the proportion of the different fatty acyl-CoAs that are produced, as different thioesterases show specificity for acyl-ACPs of different chain lengths and degree of saturation. For example, plant

FIGURE 12.1. Schematic representation of triacylglycerol biosynthesis in devel-oping seeds. ACC = acetyl-CoA caroxilase; FAS = fatty acid synthetase; SAD =stearoyl-ACP desaturase; ODS = oleoyl-phosphatidylcholine desaturase; LDS =linoleoyl-phosphatidylcholine desaturase; PC = phosphatidylcholine.

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species accumulating short-chain fatty acids posses thioesterases with sub-strate preferences for short-chain acyl-ACPs (Dehesh et al., 1996; Voelkeret al., 1997).

Free fatty acids move through the plastid membrane and are converted toCoA thioesters by acyl-CoA synthetase. The acyl-CoAs in the cytoplasmare then incorporated into lipids in the endoplasmic reticulum by acyltrans-ferases and further modifications occur (Figure 12.1). For example, 18:1-CoA is incorporated into membrane phospholipids, where the second andthird double bonds are added by the action of phospholipid desaturases.Desaturated acyl-CoAs are returned to the cytoplasmic acyl-CoA pool. Theseed storage triacylglycerols are formed by the action of three differentacyltransferases, which attach the acyl-CoAs to the three positions of theglycerol backbone (Ohlrogge, Browse, and Somerville, 1991).

Triacylglycerols are stored in specialized organelles which have been re-ferred to as oil bodies, lipid bodies, oleosomes, or spherosomes. Oil bodiesare spherical structures consisting of a core of triacylglycerol surroundedby a half-unit membrane of phospholipid (Ohlrogge, Browse, and Somer-ville, 1991). The phospholipid membrane contains specific proteins namedoleosins and caleosins (Frandsen, Mundy, and Tzen, 2001). Oleosin isthought to be important for oil body stabilization in the cytosol (Huang,1996), although neither its structure nor its function have been completelyelucidated. Little is known about caleosin, which has recently been de-scribed (Chen, Tsai, and Tzen, 1999; Naested et al., 2000). The size of theoil bodies depends on the plant species (Tzen et al., 1993).

Saturated Fatty Acids

Dietary experiments have indicated that the saturated fatty acids lauric(12:0), myristic (14:0), and palmitic (16:0) have a detrimental atherogeniceffect on human health by raising both serum total cholesterol content andlow-density lipoprotein (LDL) levels as compared with isocaloric amountsof carbohydrates (Mensink, Temme, and Hornstra, 1994). Increased serumtotal and LDL cholesterol levels are a well-known risk factor for coronaryheart disease. Conversely, neither saturated fatty acids with less than 12 car-bon atoms nor stearic acid (18:0) have been found to be hypercholesterol-emic.

The principal vegetable sources of saturated fatty acids in the world mar-ket are coconut and palm (fruit and kernel) oils, which mainly containhypercholesterolemic saturated fatty acids. Coconut and palm kernel oilmainly contain lauric acid (12:0), whereas palm oil contains a high propor-tion of palmitic acid (16:0) (Table 12.4). Dietary guidelines recommend a

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TABLE 12.4. Average composition of the principal vegetable oils and fats for major fatty acids

Fats and oils 8:0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:1(OH) 18:2 18:3 20:0 20:1 22:0 22:1 24:0Canola* 3.9 0.2 1.9 64.1 18.7 9.2 0.6 1.0 0.2 0.2Castor 1.0 1.0 3.0 90.0 4.0 trCoconut 7.8 6.7 47.6 18.1 8.8 2.6 6.2 1.6 0.1 0.1Cottonseed 0.8 24.0 0.8 2.6 19.0 52.5 tr 0.3Linseed 6.1 0.1 3.2 16.6 14.2 59.8Maize 0.1 0.2 13.0 2.5 30.5 52.0 1.0 0.5 0.2Olive 13.7 1.2 2.5 71.1 10.0 0.6 0.9Palm 0.3 1.1 45.1 0.1 4.7 38.8 9.4 0.3 0.2Palm kernel 2.5 4.0 49.0 16.0 9.0 2.0 14.0 2.0 1.0Peanut 12.5 2.5 37.0 41.0 0.3 1.2 0.7 2.5 1.0 1.3Rapeseed 3.0 1.0 16.0 14.0 10.0 1.0 6.0 tr 49.0Safflower 0.1 6.5 0.1 2.9 13.8 75.3 0.4 0.2Soybean 11.0 0.5 4.0 22.0 53.0 7.5 1.0 1.0Sunflower 0.1 5.5 0.1 4.7 19.5 68.5 0.1 0.3 0.1 0.9 0.2

Source: Data from Padley, Gunstone, and Harwood (1994) and White (1992).*Canola is the designation for rapeseed cultivars with no erucic acid in the seed oil and with low levels of glucosinolates in the oilmeal.

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reduction in the consumption of saturated fats and oils and their replace-ment by unsaturated fatty acids, which are not considered to be hypercho-lesterolemic (U.S. Department of Agriculture [USDA], 1992).

Saturated fatty acids possess advantageous technological properties forsome applications, for example, shortening and margarine manufacture.For these applications, liquid oils rich in unsaturated fatty acids must beconverted to semisolid, plastic fats by means of the hardening process,which involves the conversion of part of the unsaturated fatty acids into sat-urated fatty acids. During this process, some double bonds change theirposition and/or stereochemical configuration producing trans and posi-tional isomers, which are a major risk factor of heart disease (Willett andAscherio, 1994). In consequence, semisolid fats with a high proportion ofthe saturated fatty acids with no detrimental health effects are required. Un-fortunately, such fats are not available in the major vegetable sources (Table12.4).

Two main breeding objectives must be outlined in relation to the previ-ous discussion: the reduction in total saturated fatty acid content in edibleoils and the increase of nondetrimental saturated fatty acids in liquid oils forusing in margarine and shortening production. In the first case, lines pro-ducing oils with reduced levels of total saturated fatty acids have been de-veloped in soybean (Erickson, Wilcox, and Cavins, 1988; Fehr et al., 1991;Takagi et al., 1995; Stojšin, Alblett, et al., 1998), safflower (Velasco andFernández-Martínez, 2000), and sunflower (Miller and Vick, 1999). In thesecond case, lines with increased levels of stearic acid have been developedin soybean (Hammond and Fehr, 1983; Graef, Fehr, and Hammond, 1985;Bubeck, Fehr, and Hammond, 1989; Rahman et al., 1995) and sunflower(Osorio et al., 1995).

Unsaturated Fatty Acids

The degree of unsaturation is not only a useful criterion for fatty acidclassification, but also one of the key aspects defining the properties of fattyacids. One of the most relevant aspects to take into account is that doublebonds are the main centers of oil oxidation. The double bonds react with ox-ygen in the air in a process involving the production of free radicals, whichare implicated in a number of diseases, in tissue injuries, and in the processof aging (Shahidi, 1997). Furthermore, the lipid oxidation products are themajor source of off flavors in oils during storage (Tatum and Chow, 1992).Although intact polyunsaturated fatty acids are beneficial for human health(Horrobin, 1992), their high susceptibility to autoxidation make them unde-sirable at high levels in edible oils.

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Oleic acid (18:1, n-9) is today the preferred fatty acid for edible pur-poses, as it combines a hypocholesterolemic effect (Mensink and Katan,1989) with a much greater oxidative stability than polyunsaturated fatty ac-ids (Yodice, 1990). High concentrations of oleic acid occur naturally in ol-ive oil (Table 12.4). Oilseed breeding, however, has created additional oilsources with even higher oleic acid than olive oil. Cultivars with seed oilcharacterized by an exceptionally high oleic acid content (>75 percent)have been developed in canola (Auld et al., 1992; Rücker and Röbbelen,1997), safflower (Knowles and Mutwakil, 1963; Fernández-Martínez, delRío, and de Haro, 1993), soybean (Kinney, 1997), and sunflower (Soldatov,1976). In addition to the high nutritional value of high oleic acid oil, it alsopossesses important industrial applications (Friedt, 1988).

A series of monounsaturated fatty acids, some of them presenting func-tional groups (hidroxy, epoxy, etc.) in the carbon chain, possess importantindustrial applications but are not suitable for edible purposes because oftoxic or antinutritional effects. Some of the most relevant are erucic acid(22:1, n-9), which is mainly present in seed oils from plants of the Brassic-aceae family (e.g., rapeseed, mustards, and crambe) (Kumar and Tsunoda,1980), petroselinic acid (18:1, n-12), present in the Apiaceae (e.g., corian-der) (Knapp, 1990), vernolic acid (epoxy-18:1), found in some wild species(e.g., Vernonia spp., Euphorbia spp.) (Pascual-Villalobos et al., 1992;Thompson et al, 1994), or ricinoleic acid (hydroxy-18:1) characteristic ofthe castor bean (Canvin, 1963).

The most common polyunsaturated fatty acids in vegetable oils arelinoleic acid (18:2, n-6) and alpha-linolenic acid (18:3, n-3). Both fatty ac-ids are of great value from a nutritional point of view, as they are essentialfatty acids. This means that they must be included in the diet because thehuman body is not able to manufacture them (Horrobin, 1992). Essentialfatty acids have an important structural function in the cell membranes andalso play a crucial role as precursors of metabolic regulators and other im-portant metabolites such as prostaglandins (Vles and Gottenbos, 1989). Inaddition, polyunsaturated fatty acids have a hypocholesterolemic effect inhumans (Chan, Bruce, and McDonald, 1991). Despite their high nutritionalvalue, polyunsaturated fatty acids are undesired in edible oils because oftheir high susceptibility to oxidation during processes such as storage orheating. Alpha-linolenic acid, with three double bonds in the molecule, ismuch more susceptible to oxidation than linoleic acid, which possesses twodouble bonds.

Alpha-linolenic acid is present at high or relatively high proportions insome commercial vegetable oils, especially in linseed (60 percent of totalfatty acids, see Table 12.4), rapeseed and canola (about 10 percent of total fattyacids), and soybean (about 8 percent of total fatty acids). Linseed oil is not

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suitable for edible purposes because of its high alpha-linolenic acid concen-tration (Frankel, 1991). However, it is precisely this characteristic thatmakes linseed oil unsurpassed as a drying oil for use in paints, varnishes,printing inks, etc. (McHughen, 1992). Successful breeding through muta-genesis led to the development of linseed mutants with seed oil containingless than 2 percent alpha-linolenic acid (Green, 1986; Rowland and Bhatty,1991). Such an oil is of great value for edible purposes (Bickert, Lühs, andFriedt, 1994). The utilization of mutagenesis has also enabled important re-ductions of linolenic acid content in rapeseed/canola (Rakow, 1973; Röb-belen and Nitsch, 1975; Wong and Swanson, 1991; Auld et al., 1992; Hitzet al., 1995) and soybean (Hammond and Fehr, 1983; Wilcox, Cavins, andNielsen, 1984; Takagi et al., 1990; Hitz et al., 1995; Stojšin, Luzzi, et al.,1998b).

Gamma-linolenic acid is a polyunsaturated fatty acid that attracts muchinterest because of its many health benefits (Fan and Chapkin, 1998). Cur-rent commercial sources of gamma-linolenic acid for the pharmaceuticalindustry are evening primrose (Oenothera biennis L.) and borage (Boragoofficinalis L.). Other interesting sources of this fatty acid are the fruits ofcurrants and gooseberries (Ribes spp.), also rich in antioxidant compounds,which increases the biological value of gamma-linolenic acid (Goffmanand Galletti, 2001).

Triacylglycerol Structure in Vegetable Oils

The functional and nutritional characteristics of an oil are affected notonly by the fatty acid composition, but also by the triacylglycerol structure,i.e., the position of the fatty acids on the glycerol backbone (Reske,Siebrecht, and Hazebroek, 1997). The stereochemical positions of the threefatty acids in the glycerol molecule are designated sn-1, sn-2, and sn-3 (Fig-ure 12.2). The distribution of fatty acids within the triacylglycerol moleculeis not random. Initial studies on seed oils concluded that saturated fatty ac-ids were virtually excluded from the sn-2 position and randomly distributedbetween sn-1 and sn-3 positions (van der Wal, 1960). Later studies, how-ever, demonstrated that triacylglycerol stereospecificity was more complexthan initially anticipated. In safflower seeds, the acylation of position sn-1has selectivity for saturated fatty acids, whereas position sn-3 has no selec-tivity (Ichihara and Noda, 1982). Conversely, saturated fatty acids in sun-flower showed preference for the sn-3 over the sn-1 position (Reske,Siebrecht, and Hazebroek, 1997).

Fatty acid stereospecificity within the triacylglycerol molecule plays anessential role in lipid nutritional value, as the absorption rates of fatty acids

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depend on the location of fatty acids in the triacylglycerol (Small, 1991). Inthe case of atherogenic fatty acids (some of the saturated fatty acids, seesection discussing fatty acids in this chapter), their absorption rate is higherwhen they are sterified at the central sn-2 triacylglycerol position than whenthey are at the external sn-1 and sn-3 positions (Bracco, 1994). Thus, vege-table oils containing a high proportion of saturated fatty acids at the sn-2position are considered to be more atherogenic than those having similar to-tal saturated fatty acid content but distributed in the external positions(Renaud, Ruf, and Petithory, 1995). Palm oil, widely used in food products,contains approximately 10 percent saturated fatty acids at the sn-2 position(Padley, Gunstone, and Harwood, 1994). Recently, mutant lines of sun-flower and soybean with increased levels of saturated fatty acids almost ex-clusively at the sn-1, 3 positions have been developed (Álvarez-Ortegaet al., 1997; Reske, Siebrecht, and Hazebroek, 1997). Besides the advan-tages derived from the positional distribution of saturated fatty acids, seedoils from these mutants possess adequate technological properties for mar-garine and other solid-fat substitute production without need of detrimentalphysical transformations such as hydrogenation or tranesterification (Listet al., 1996; Kinney, 1999).

Natural Antioxidants in Vegetable Oils

Oxidative processes occur both in vitro and in vivo. Autoxidation oflipids during storage (in vitro) is one of the main factors diminishing food

R

O

C CO

H2

H2

C

O

O C R

O

C ROC

H

sn-1

sn-2

sn-3

FIGURE 12.2. Structure of a triacylglycerol molecule. sn-1, sn-2, and sn-3 referto the carbon numbers of the glycerol; sn-2 is a chiral center. R represents fattyacids.

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quality. This process affects not only fats and oils, but also feeds and foodscontaining them. The consequence of oxidation is the development of un-pleasant tastes and odors (rancidity) and degradation of functional and nu-tritional properties (St. Angelo, 1996; Crapiste, Brevedan, and Carelli,1999). Oxidation also occurs in the human body (in vivo), promoting theformation of reactive oxygen and nitrogen species (free radicals), whichcause damage to DNA, lipids, proteins, and other biomolecules. Diet-derived antioxidants are of paramount importance in maintaining health, asendogenous antioxidant defenses are inadequate to prevent damage com-pletely (Halliwell, 1996). Vegetable oils are one of the most importantsources of natural antioxidants, the most important being described in thefollowing sections.

Chromanols

Chromanols consist of a chroman head with two rings, one phenolic andone heterocyclic, the latter substituted with a phytyl tail (Kamal-Eldin andÅppelqvist, 1996). The most important chromanols are tocopherols, withsaturated phytyl tails, tocotrienols, having unsaturated phytyl tails, andplastochromanol-8, with a saturated phytyl tail longer than that of tocoph-erols. Tocopherols and tocotrienols include mono ( -), di ( - or -), andtrimethyl ( -) tocol derivatives (Figure 12.3).

FIGURE 12.3. Chemical formulas of (A) tocopherols and (B) tocotrienols. Me =methyl groups.

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The chromanols exhibit antioxidant activity both in vivo and in vitro. Invivo they exert vitamin E activity, protecting cellular membrane lipidsagainst oxidative damage (Muggli, 1994). In vitro they inhibit lipid oxida-tion in oils and fats, as well as in foods and feeds containing them (Kamal-Eldin and Åppelqvist, 1996). The biologically most active chromanol formis -tocopherol (Traber and Sies, 1996). According to Padley, Gunstone,and Harwood (1994), the greatest vitamin E effect is exhibited by -tocoph-erol (relative activity 100), followed by -tocopherol (50), -tocotrienol (30),and -tocopherol (10). Conversely, the best in vitro antioxidant activity ranksin the order -tocopherol (relative antioxidant activity 100), -tocopherol(68), -tocopherol (64), and -tocopherol (35) (Pongracz, Weiser, and Mat-zinger, 1995). The in vitro antioxidant activities of tocotrienols are un-known, whereas it has recently been shown that plastochromanol-8 is amore powerful antioxidant than -tocopherol (Olejnik, Gogolewski, andNogala-Kalucka, 1997)

The tocopherol derivatives are the predominant chromanol form in vege-table oils. The most relevant exceptions are palm oil, which in addition totocopherols contains large amounts of tocotrienols (Padley, Gunstone, andHarwood, 1994), and linseed oil, which contains both tocopherol deriva-tives and plastochromanol-8 (Velasco and Goffman, 2000). The averagechromanol composition of vegetable oils is given in Table 12.5.

Because of their beneficial in vivo and in vitro action, the increase of to-tal chromanol content of vegetable oils is an important objective in oilseedbreeding. Also, since the chromanol derivatives differ in their relative invivo and in vitro activity, the modification of the chromanol profile for spe-cific end uses of the modified oil or fat is an important breeding goal. Thus,Shintani and Dellapenna (1988) focused on increasing the proportion of

-tocopherol in vegetable oils as a way of increasing their vitamin E activ-ity. They followed a biotechnological approach, based on overexpression of-tocopherol methyltransferase (TMT), to convert naturally occurring-tocopherol of Arabidopsis seed oil into -tocopherol. The resulting oil

from Arabidopsis lines overexpressing -TMT was ninefold that of the oilfrom the wild-type line. Conversely, Demurin, Skoric, and Karlovic (1996)attempted to increase the concentration of -tocopherol content in sun-flower oil with the aim of improving oil oxidative stability. By evaluatingthe existing variability in sunflower germplasm, they identified and se-lected lines with seed oils containing 50 percent -tocopherol and 50 per-cent -tocopherol, and lines producing 95 percent -tocopherol, in compari-son with standard lines characterized by 95 percent -tocopherol.

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Carotenoids

Carotenoids are 40-carbon polyunsaturated hydrocarbons (carotenes)and their oxygenated derivatives (xanthophylls). Carotenes are either linearor cyclized at one or both ends of the molecule (Goodwin, 1980). Similar tochromanols, they play an important role in vivo as source of provitamin A,as well as in vitro, protecting oils from oxidation (Henry, Catignani, andSchwartz, 1998; Hirschberg, 1999). -carotene is the most nutritionally ac-tive carotene as provitamin A (Ong and Choo, 1997). Their main in vivoprotective effect has traditionally been attributed to antioxidant action(Palozza and Krinsky, 1992; Miller et al., 1996). Other benefits of caroten-oids such as conversion to retinoids or effects on cell communication havealso been described (Halliwell, 1996). The in vitro activity of carotenoidsseems to be related to the inhibition of photooxidation, acting as a filter forlight of short wavelengths (Warner and Frankel, 1987).

Palm oil is the richest oil source of carotenoids, with a concentration of500 to 700 ppm (Ong and Choo, 1997). The concentration of carotenoids inseed oils is considerably lower (Uppström, 1995). Some experiments di-

TABLE 12.5. Chromanol content (mg/kg) of oils and fats

Fats and oilsTocopherols Tocotrienols

P-8a

Canola 202 65 490 9 –b – – –Castor 28 29 111 310 – – – –Coconut – – – 4 20 – – –Cottonseed 338 17 429 3 – – – –Linseed 4 – 407 – – – – 142Maize 282 54 1034 54 – – – –Olive 93 – 7 – – – – –Palm 89 – 18 – 128 323 72 –Palm kernel 62 – – – – – – –Peanut 178 9 213 8 – – – –Safflower 477 – 44 10 – – – –Soybean 100 8 1021 421 – – – –Sunflower 670 27 11 – – – – –

Source: Data from Padley, Gunstone, and Harwood, 1994, except for linseedfrom Velasco and Goffman, 2000.aPlastochromanol-8bIndicates absence of compound in oil

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rected to the manipulation of carotenoid production in plants through meta-bolic engineering have already been conducted (Hirschberg, 1999), thoughso far no attempt has been made to alter carotenoid contents of vegetableoils.

Phenolic Compounds

Polar phenolic compounds are important natural antioxidants present inolive oil, especially in extra virgin oil, which is obtained from the fruitmesocarp by mechanical pressing (Tsimidou, Papadoupoulos, and Boskpu,1992). They include a wide variety of simple phenols (e.g., hydroxytyrosol,tyrosol), aldehydic secoiridoids (e.g., oleuropein and derivatives), flavo-noids, and lignans (Owen et al., 2000). According to Aparicio and col-leagues (1999), phenolic compounds are the main contributors to olive oilstability. Among them, hydroxytyrosol, oleuropein, and caffeic acid seemto be the most powerful antioxidants (Saija et al., 1998; De la Puerta,Gutierrez, and Hoult, 1999). Nevertheless, many of the phenolics present inolive oil have not yet been completely identified (Shukla, Wanasundara,and Shahidi, 1997).

In general, seed oils are devoid of phenolic compounds. One exception issesame oil, which contains a powerful antioxidant, sesamol, as well as sev-eral bisfuranyl lignans (Potterat, 1997).

Phytosterols

Phytosterols or plant sterols are essential components of the membranes,playing an important role in the control of membrane fluidity and perme-ability as well as in signal transduction. Their role in plant cells is similar tothat of cholesterol in mammalian cells (Piironen et al., 2000). Chemically,they are steroid alcohols (triterpenes) synthesized from squalene in theisoprenoid pathway (Benveniste, 1986). Vegetable oils are the richest natu-ral sources of plant sterols. Among them, the highest contents are found inmaize and rapeseed (Rossell and Pritchard, 1991). The predominant sterolsin vegetable oils belong to the 4-desmethyl sterol class, which contributesmore than 85 percent of total sterols. Within this group, sitosterol (usuallyabove 50 percent of 4-desmethyl sterols), campesterol, sigmasterol, 5-avenasterol, 7-avenasterol, and 7-stigmastenol are the most significant(Piironen et al., 2000). Brassicasterol is a sterol characteristic of the Bras-sicaceae family and therefore is present in rapeseed/canola oils (Uppström,1995). Phytosterols of vegetable oils occur mainly as free sterols and estersof fatty acids, especially as esters of oleic and linoleic acids (Piironen et al.,

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2000). Saturated plant sterols (stanols) occur in low amounts in vegetableoils (Dutta and Åppelqvist, 1996).

Plant sterols and stanols contained in vegetable oils lower total and LDLserum cholesterol in humans by inhibiting cholesterol absorption from theintestine. Stanols have greater potential to lower cholesterol than sterols be-cause they are virtually unabsorbable (Nguyen, 1999). Plant sterol andstanol esters are currently being incorporated into food products such asmargarines as a dietary ingredient for lowering serum cholesterol (Miet-tinen et al., 1995).

Oil Components with Anticarcinogenic Activity

As a general rule, all oil components with antioxidant action are of greatvalue in cancer prevention, as free radicals produced through oxidative pro-cesses are directly implicated in carcinogenesis (Borek, 1993). In addition,some oil components without a marked antioxidant role have an anticancerprotective effect through other mechanisms of action. One of the most rele-vant examples is squalene, a triterpene of the isoprenoid pathway (Benven-iste, 1986). In most vegetable oils, squalene is an intermediary in the syn-thesis of phytosterols and its final concentration in the oil is low. In olive oil,however, there is an important accumulation of squalene (Kiritsakis, 1987),which has been found to have very weak antioxidant activity (Psomiadouand Tsimidou, 1999). Its chemopreventive efficacy seems to be through astrong inhibitory activity of certain enzymes implicated in oncogene activa-tion (Newmark, 1999).

MEAL QUALITY

Oilseed meals are extensively used as protein supplements for use in ani-mal feeds, as approximately between 20 and 50 percent of weight of mealsis protein. Some oilmeal is further processed to produce concentrates (with50 to 60 percent of crude protein) or isolates (nearly pure protein) for use inhuman food (Bell, 1989). Oilmeals are mainly valued for low fiber content,high protein content of good quality, and absence or low presence of toxicand antinutritional compounds.

Fiber Content

Fiber is not a homogenous chemical entity. It refers to the carbohydratesthat are not truly digested by the animal and therefore do not contribute en-

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ergy when consumed. They include cellulose, hemicellulose, pectins, gums,mucilages, and lignin-hemicellulose complexes (Vohra, 1989).

Fiber is predominantly associated with the seed hull. Some oilseeds, par-ticularly sunflower and safflower, are characterized by a high hull propor-tion. In the case of sunflower, hull percentage among genotypes may varyfrom 10 to 60 percent of the total achene weight (Miller and Fick, 1997). Insafflower, hull content ranges from 20 percent in reduced-hull genotypes toabout 45 percent in white-hull types (Fernández-Martínez, 1997). In theseoilseeds, dehulling is necessary to render meals useful as protein supple-ments (Bell, 1989). In sunflower, it has been shown that genetic variabilityfor hullability or facility for dehulling exists (Denis, Domínguez, and Vear,1994). Other oilseeds contain much lower hull contents, for example, be-tween 15 and 20 percent of seed weight in rapeseed (Niewiadomski, 1990)and 7 percent to 8 percent of seed weight in soybean (Mounts, Wolf, andMartinez, 1987). In these cases the oilmeals are commercially available ei-ther with or without hulls or may be partially dehulled (Bell, 1989).

In some cases it has been possible to reduce fiber content by selecting forlower hull content. In rapeseed/canola, yellow seeds are characterized bythinner seed coats than dark seeds, which is associated with about 4 percentlower fiber content (Stringam, McGregor, and Pawlowski, 1974). This facthas encouraged breeding for yellow-seeded types as a means of improvingthe nutritional value of the meal (Baetzel, Friedt, and Lühs, 2000). In saf-flower, the identification of a thin-hulled mutant (Ebert and Knowles, 1966)allowed a reduction of crude fiber content from about 30 to 11 percent of thetotal seed (achene) weight (Weiss, 1983).

Protein Content and Quality

The oilmeals obtained after oil extraction from oilseeds contain high lev-els of protein, from about 20 percent in palm kernel meal to about 45 to 50percent in soybean meal. Most oilseeds yield a defatted meal with about 35to 50 percent crude protein (Table 12.2).

Proteins are polymers of amino acids. The amino acids are a group of pri-mary amines that contain a central carbon atom to which are attached a hy-drogen atom, an amino group (NH2), and a carboxyl group (COOH). Pro-teins of oilseeds can be divided into three functional groups: (1) storageproteins with no enzymatic activity, which are the most abundant; (2) pro-teins having a structural function; and (3) enzymes (Niewiadomski, 1990).Another generalized classification of proteins is based on their solubility invarious solvents. In this system, the four classical types of proteins are albu-mins, globulins, prolamins, and glutenins (Osborne, 1924). The globulins

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represent the major storage proteins in all oilseeds (Bell, 1989), accountingfor about 90 percent of the seed protein in soybean (Wilson, 1987), 70 per-cent in rapeseed/canola (Uppström, 1995), and 60 percent in sunflower(Dorrell and Vick, 1997).

As long as the proportion of hull remains constant, oil and protein con-tents are negatively correlated (Röbbelen, 1981). In consequence, selectionfor high protein usually results in lower oil content. Because of the negativecorrelation between both traits, some authors have suggested separate de-velopment of high-oil and high-protein cultivars (Röbbelen, 1981). Others,however, recommended conducting selection for the sum of oil and proteinwhile maintaining acceptable oil content, since the latter has a higher pricein the market (Jímenez et al., 1985; Miller and Fick, 1997).

The quality of the protein has to be evaluated in terms of the nutritionalbalance of the absorbed amino acids. The amino acids of major interest arearginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine,threonine, tryptophan, and valine, which are essential amino acids; i.e., theycannot be synthesized by the human body and have to be incorporatedthrough the diet. The amino acids cysteine, tyrosine, and glutamic acid arenot essential but can partially satisfy the need for essential amino acids(Bell, 1989).

Nutritionally, most oilseed meals are deficient in some amino acids (Table12.2). Soybean meal has a limited content of sulfur amino acids methionineand cysteine, although it has an adequate content of the other essentialamino acids. It is worth noting the high lysine content of soybean meal,which complements the low lysine content of cereals (Bell, 1989). In addi-tion to a limited content in sulfur amino acids, the other important legumeoilseed, peanut, is also characterized by a low lysine content. The proteinsof the nonlegume oilseeds are nutritionally adequate in sulfur amino acidsand, with the exception of rapeseed/canola, nutritionally inadequate inlysine (Norton, 1989). The seed protein of rapeseed/canola has the best bal-anced amino acid composition of oilseeds, also comparing favorably withcereals (Rosa, 1999).

Toxic and Antinutritional Compounds in Oilmeals

Plants produce and accumulate potentially toxic compounds as a chemi-cal protection against herbivores (Ågren and Schemske, 1993). In the caseof oilseeds, most of the compounds with toxic or antinutritional propertiesremain in the meal after oil extraction, considerably reducing its value forhuman food and animal feed. Toxic compounds may have serious deleteri-ous effects on both livestock and humans. Antinutritional compounds re-

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duce the nutritive value of the feed, mainly by negatively affecting palat-ability, digestibility, or both. Most of the compounds or classes of com-pounds with detrimental properties present in oilseeds are toxic if ingestedat high concentrations, but their effects at low concentrations are morelikely to be antinutritional (Griffiths, Birch, and Hillman, 1998).

Some classes of antinutritional compounds are widely distributed in oil-seeds (e.g., phytates, phenolics) while others are specific to certain plantfamilies (e.g., glucosinolates in the Brassicaceae). Some of the most rele-vant are briefly described as follows.

Phytates

Phytic acid (myoinositol 1, 2, 3, 4, 5, 6-hexakis-dihydrogen phosphate)is a major component of cereals and oilseeds, constituting between 1 and 3percent of the total seed weight. In oilseeds, phytic acid usually occurs as amixture of calcium, magnesium, and potassium salts (phytates), in cristal-oid-type globoids in the cells of the radicle and the cotyledon (Yiu, Alto-saar, and Fulcher, 1983). Physiologically, phytic acid plays an importantfunction as a primary reserve of energy, phosphorus, and myoinositol in theseed (Graf, 1983). Between 50 and 80 percent of the phosphorus of oilseedsis stored in the form of phytic acid (Lolas, Palamidis, and Markakis, 1976).This phosphorus is nutritionally unavailable to nonruminant livestock (Erd-man, 1979).

Phytic acid is a strong chelating agent that can bind metal ions, reducingthe availability of calcium, iron, magnesium, zinc, and other trace elements(Oberleas, Muhrer, and O’Dell, 1966). In addition, phytates form com-plexes with amino acids, reducing digestibility and amino acid availability(Thompson, 1990). These antinutritional properties limit the use of the oil-seed protein for animal feed. In human nutrition, however, several benefi-cial effects have been reported. Phytic acid is believed to have a marked invivo antioxidant effect, to decrease the risk of iron-mediated colon cancer,and to lower serum cholesterol and triglycerides (Martínez et al., 1995;Greiner and Jany, 1996).

Among oilseeds, rapeseed/canola contains the highest concentration ofphytic acid. Matthäus, Lösing, and Fiebig (1995) reported a range from 2.0to 4.0 g/100 g seed in comparison with 2.5 to 2.6 g/100 g in linseed, 1.9g/100 g in peanut, 1.2 to 1.7 g/100 g in soybean, and 1.9 g/100 g in sun-flower. Because of the chelating action of phytic acid, oilmeals for animalfeed are often supplemented with the enzymes phytase and acid phos-phatase, which improves their digestibility and increases the bioavailabilityof phosphorus and metal ions (Zyla and Korelski, 1993; Aldeola, 1995).

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Another strategy is to reduce the ratio of phytate phosphorus to inorganicphosphorus in the seed by plant breeding. This has recently been achievedin soybean by Wilcox and colleagues (2000), who developed two mutantswith considerably increased proportion of inorganic phosphorus in relationto phytate phosphorus.

Phenolics

Phenolic compounds are common in most oilseeds. They exert a detri-mental effect on meal quality by interacting with amino acids, denaturingproteins, and inhibiting enzymes, thus lowering the nutritional value of themeal for animal feed (Sozulski, 1979). Phenolic compounds also limit theutilization of the meal as a source of human food-grade protein becausethey confer undesired color, bitter taste, and/or astringency to oilseed pro-tein products (Naczk et al., 1998).

The content of phenolics in rapeseed/canola is much higher than in otheroilseeds, about ten times that in peanut and cottonseed and about 30 timesthat in soybean (Shahidi and Naczk, 1992). The most important phenoliccompound in rapeseed/canola seeds is sinapine, the choline ester of sinapicacid, which represents from 5.0 to 17.7 g•kg–1 total seed weight (Velascoand Möllers, 1998). Another important phenolic compound is chlorogenicacid, which is responsible for the production of yellow-green coloration fol-lowing oxidation in sunflower meal. The presence of chlorogenic acid lim-its the broad use of sunflower meal for human consumption (Dorrell andVick, 1997). Both sinapine and chlorogenic acid are predominantly presentin the seed kernels; therefore, the dehulling of the seeds scarcely reducesthe presence of these phenolics in the meal (Uppström, 1995; Pedrosa et al.,2000).

Tannins are complex phenolic compounds (polyphenolic compounds)that form complexes with proteins and reduce their availability to animals.They are present in variable proportions in most oilseeds. Matthäus (1997)reported a variation from 0.04 mg/g in soybean to 3.8 mg/g in rapeseed.Peanuts, not included in the mentioned evaluation, also have a significantamount of tannins (Bell, 1989). In peanuts, tannins are mainly concentratedin the testa, which is usually removed to improve energy digestibility of themeal (Weiss, 1983).

Glucosinolates

Glucosinolates (GSLs) are a family of secondary plant metabolites par-ticularly abundant in seeds and green tissues of the family Brassicaceae

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(Kjaer, 1976). They consist of a thioglucoside linked to a variety of sidechains which are usually amino acid derivatives (Höglund et al., 1991).More than 100 different GSLs showing different side chain structure havebeen identified in the plant kingdom, although only around 15 or 16 occurin significant amounts in the genus Brassica, to which the oilseeds rapeseedand canola belong (Rosa, 1999). Both the GSLs and their degradation prod-ucts are associated with antinutritive and toxic effects, limiting the useful-ness of seeds and seed meals for human and animal feed (Sørensen, 1990).

Traditional rapeseed cultivars contained high levels of glucosinolates,about 110 to 150 mol/g seed. The discovery in the middle 1960s that thePolish variety ‘Bronowski’ contained much lower glucosinolate content,about 10 to 12 moles/g seed (Josefsson and Åppelqvist, 1968; Downey,Craig, and Youngs, 1969), opened up the development of rapeseed cultivarswhich combined the previously developed zero erucic acid trait (Stefans-son, Hougen, and Downey, 1961) with very low glucosinolate levels. Cano-la was the name adopted by the rapeseed industry in Canada in 1978 to des-ignate rapeseed (Brassica napus and B. rapa) cultivars with less than 1 percenterucic acid in the seed oil and a glucosinolate content below 30 mol/g oil-extracted, air-dried meal (Vaisey-Genser and Eskin, 1987). The drastic re-duction of glucosinolates took place in the seeds but not vegetative tissues,which maintained similar high glucosinolate content as the traditionalcultivars. This fact was of great value for the productive potential of the newlow-glucosinolate cultivars, as glucosinolates have an important protectiveeffect against pests and diseases (Mithen, 1992).

Other Antinutritional Factors

Among the most important antinutritional compounds present in soy-bean seeds are trypsin inhibitors. Trypsin inhibitors are types of protease in-hibitors, which are proteins found in virtually all legume species (Lienerand Kakade, 1980). Soybean trypsin inhibitors cause pancreatic lesions,particularly hypertrophy and hyperplasia, in animals fed with raw soybeanmeal (Liener et al., 1985). The trypsin inhibitors are heat labile, being inac-tivated during the toasting phase of meal production. Toasted meals do not,therefore, cause problems to animals fed with soybean meal (Rackis, Wolf,and Baker, 1985).

Cottonseed meal contains gossypol, a polyphenolic compound presentin the pigment glands of both the vegetative tissues and the seeds of cotton(Kohel, 1989). Gossypol is toxic to monogastric animals and also causesdiscoloration in foods (Vroh-Bi et al., 1999). The identification of glandlesstypes of cotton resulted in a great improvement of meal quality (McMichael,

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1960) but also in a greater susceptibility to insect attack (Calhoun, 1997). Inconsequence, breeding efforts to develop glanded-plant, glandless-seedcottonseed cultivars are under way (Vroh-Bi et al., 1999).

Castor seeds contain two highly toxic endosperm proteins, ricin andRicinus communis agglutin, which limit the utilization of castor meal foranimal nutrition (Pinkerton et al., 1999). Seeds of linseed contain cyano-genic glycosides which, upon hydrolysis by enzymatic action, release hy-drogen cyanide (HCN), a powerful inhibitor of the respiratory enzymecytochrome oxidase. Nevertheless, the enzyme responsible for the hydroly-sis of the cyanogenic glycosides is inactivated by heat during hot-pressingoil extraction (Oomah, Mazza, and Kenaschuk, 1992; Shahidi and Wana-sundara, 1997).

BREEDING AND PRODUCTION STRATEGIES

Ultimately, production of oil crops aims at maximizing the profit fromthe harvest. Most oil crops produce two main products: oil and meal. Inmost cases the biggest profit is obtained from the oil, while oilmeal makes asecondary contribution to the overall economic value of the harvest. Thereare exceptions to this rule, the most representative being soybean products.Soybean seeds contain a relatively low oil content as compared with theother oil crops. One ton of soybean seed yields approximately 180 kg oiland 800 kg meal. The ratio of the sale value of meal to that of the oil changesfrequently depending on the situation in particular markets (Hatje, 1989).

Increasing oil yield of oilseeds is achieved by increasing seed yieldand/or increasing the oil content of the seed. Oil content depends on boththe percentage of hull and the oil concentration in the kernel. In most cases,significant increases of seed oil content have been achieved by a reductionin hull percentage. In sunflower, it has been estimated that about two-thirdsof the increase in achene oil content occurred during selection for this traitresulted from reduction in hull percentage, and about one-third from an in-crease in kernel oil content (Fick and Miller, 1997). Oil content in the seedkernel is considered to be a quantitative trait strongly influenced by the en-vironment, although it shows a relatively high heritability in comparisonwith other quantitative traits such as yield (Grami, Baker, and Stefansson,1977; Röbbelen, 1990; Miller and Fick, 1997). Consequently, plant breed-ers have been able to increase seed kernel oil content by changing theoil:protein:carbohydrate ratios in favor of oil by using conventional breed-ing methods (Murphy, 1995). Further increases using classical breeding ap-proaches are becoming progressively more difficult and efforts to improve

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oil content by biotechnological means are under way (Töpfer, Martini, andSchell, 1995; Zou et al., 1997; Martini and Schulte, 1998).

Traditionally, the fatty acid composition of the oil has been considered tobe the main factor defining oil quality. Therefore, great breeding effortshave been devoted to its modification for special purposes, including bothfood and nonfood uses of the oils. The concentration of a particular fattyacid in the seed oil is a qualitative trait governed by a reduced number ofmajor genes. With few exceptions in which significant maternal effectshave been reported, fatty acid concentration is determined by the genotypeof the developing embryo (Velasco, Pérez-Vich, and Fernández-Martínez,1999). This fact has considerably facilitated breeding for modified fattyacid composition, as early selection on single seeds is as effective as selec-tion on single plants (Röbbelen, 1990). Based on this advantage, the half-seed technique for nondestructive selection for seed oil fatty acid composi-tion was developed by Downey and Harvey (1963) and extensively used foroilseed breeding over the past forty years.

Environmental factors, especially temperature during seed development,influence the fatty acid composition of the oil. Canvin (1965) demonstratedthat, in general, the level of unsaturation of the oil decreases as temperatureincreases. In sunflower, this effect has been extensively studied for thedesaturation step from oleic acid (18:1) to linoleic acid (18:2). It has beenconcluded that temperature exerts a multiple effect on oleic acid desatur-ation, affecting the availability of substrate (oleate), the activity of themicrosomal oleate desaturase (FAD2) enzyme (Garcés, Sarmiento, andMancha, 1992), and the availability of oxygen, which in turn is also in-volved in the regulation of the enzyme (Martínez-Rivas, García-Díaz, andMancha, 2000). As a result, higher temperatures promote higher levels ofoleic acid content in the oil. This fact has traditionally been used as an im-portant criterion for commercial production of sunflower in the UnitedStates, where oil obtained from warm regions has been used for specificmarkets requiring higher levels of oleic acid (Robertson, Morrison, andWilson, 1978). The influence of temperature on the fatty acid profile of theoil is genotype dependent. A remarkable example is the high oleic acid sun-flower mutant developed by Soldatov (1976), which is much less sensitiveto temperature fluctuations than standard sunflower. In an experiment con-ducted in a growth chamber, Fernández-Martínez and colleagues (1986)found a range of variation for oleic acid between 38 and 62 percent in stan-dard sunflower compared with 91 to 95 percent in high-oleic acid sun-flower. The commercial production of an oil crop having a specific fattyacid profile requires its stable expression over environments, which repre-sents one of the major goals in oilseed breeding.

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Major achievements in the modification of the fatty acid composition ofseed oil have been accomplished in all important oilseeds. Particularly suc-cessful has been the induction of mutations with physical or chemicalmutagenizing agents, which has produced a wide variation in fatty acid pro-file in rapeseed/canola, sunflower, soybean, and linseed (see review inVelasco, Pérez-Vich, and Fernández-Martínez, 1999). In recent years, how-ever, major attention has been paid to the utilization of genetic engineeringfor the modification of the fatty acid biosynthetic pathway (Kinney, 1999).

The elimination of potentially toxic and/or antinutritional factors hasbeen the main objective in breeding for meal quality in most oilseed crops.One of the most spectacular achievements has been the improvement inmeal quality of old rapeseed cultivars by drastically reducing the gluco-sinolate content. Such a modification resulted in a change in status of thecrop, from low-quality to high-quality meal for animal feed (Buzza, 1995).As described for oil quality, breeding efforts on meal quality are shifting to-ward a greater weight of biotechnological approaches. Thus, a number ofreports have described the successful application of genetic transformationfor the modification of the total seed protein content (Kohno-Murase et al.,1994), amino acid composition (Clercq et al., 1990; Altenbach et al., 1992;Kohno-Murase et al., 1995), tocopherol composition (Shintani and Della-penna, 1998), and antinutritional compounds (Chavedej et al., 1994).

A major problem that oilseed breeders have had to confront is that eachchange in the oil or meal quality has initially been associated with reduc-tions in seed and oil yields, in comparison with earlier quality forms. Thereason for this cannot be attributed to physiological constraints but to atransfer of selection intensity from yield to quality traits (Becker, Löptien,and Röbbelen, 1999). However, cultivars with improved oil or meal qualityand similar or even better agronomic performance than earlier quality cul-tivars can be developed by maintaining an adequate selection intensity onyield. Current sunflower cultivars with high oleic acid content (Fernández-Martínez, Muñoz, and Gómez-Arnau, 1993), canola cultivars exhibiting asimultaneous reduction of erucic acid and glucosinolates (Röbbelen andThies, 1980), and linseed cultivars with low linolenic acid content (Scarth,Mcvetty, and Rimmer, 1997) are illustrative examples.

Oil crops, particularly oilseeds, have traditionally been at the forefront ofplant breeding. This leading position has been augmented with the adventof the biotechnological revolution. In the next several years novel breedingtools derived from biotechnology and molecular genetics will be called onto play an increasingly important role. Nevertheless, effective improvementof grain quality in oil crops will necessarily require the adequate integrationof such novel tools with traditional breeding approaches.

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position in the Brassicaceae by expression of a yeast sn-2 acyltransferase gene.Plant Journal 9: 909-923.

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Chapter 13

The Malting Quality of BarleyThe Malting Quality of Barley

Roxana SavinValeria S. Passarella

José Luis Molina-Cano

INTRODUCTION

Importance of Grain Quality

Grain quality comprises a group of characteristics that collectively deter-mine the usefulness of the harvested grains for a particular end use. Fre-quently it is regarded by both breeders and producers to be as important asyield. Not only are these characteristics the reason why only a few plantspecies are used to satisfy most human requirements for food and fiber(Slafer and Satorre, 1999), but also their importance for grain trading hasbeen increasing in recent decades (Wrigley, 1994). Therefore, to breed andmanage grain crops to achieve a certain quality standard and to be able topredict the quality of a particular crop is rather important. Achievement ofthis objective is dependent upon our knowledge of the major factors thatcould modify grain composition and, consequently, its quality.

As grain markets have become more specialized, there is growing pres-sure on farmers to produce grains with greater uniformity and with certaincharacteristics (Wrigley, 1994). Appropriate husbandry to obtain grainswith high and stable quality will likely be of increasing importance inachieving economic benefits. It is well known that grain quality may bemodified by environment or crop management techniques. However, thestrategies and tools required to produce grains with certain quality charac-teristics are not as well established as those for achieving high yields.Within this context, it has become increasingly important to improve our

We wish to thank Stuart Swanston (Scottish Crop Research Institute) and GustavoSlafer (University of Buenos Aires) for critical reading and suggestions to this chapter.We are deeply indebted to Sandy MacGregor (Canadian Grain Commission) who gener-ously allowed us to use his original micrographs reproduced in Figure 13.2.

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understanding of the factors that determine grain quality (Gooding andDavies, 1997).

Malting and Brewing Processes

The essential aim of brewing is the conversion of cereal starch into alco-hol to make a palatable beverage. Two processes are involved: the starch isfirst converted to soluble sugars by amylolytic enzymes, and second, thesugars are fermented to alcohol by enzymes present in yeast (Kent andEvers, 1994). Only the key aspects of the malting and brewing processes aresummarized here, to provide the reader with a general framework to under-stand the subsequent sections of this chapter. Further and more comprehen-sive treatment of the topic can be found in Cook (1962), Briggs (1978),Pollock (1979), Briggs and colleagues (1986), Hough and colleagues (1987),Palmer (1989), Moll (1991), MacGregor and Bhatty (1993), Hardwick(1995), Lewis and Young (1995), and Kunze (1996).

Malting can be defined as the commercial exploitation of those pro-cesses that lead to germination (Sparrow, 1970, cited by Swanston andEllis, 2002). The malting process commences with the steeping of barley inwater to achieve a moisture level sufficient to activate metabolism in theembryonic and aleurone tissues, leading in turn to the development ofhydrolytic enzymes (Figure 13.1) (Bamforth and Barclay, 1993). Moistureuptake into the starchy endosperm is also critical before the food reserves ofthat tissue can be mobilized through the action of enzymes during the ger-mination process. In this mobilization phase, referred to as modification,the cell walls and protein matrix of the starchy endosperm are degraded, ex-posing the starch granules and rendering the grain friable and readilymilled. After a period of germination sufficient to achieve an even modifi-cation, the green malt is dried and kilned to arrest germination and stabilizethe malt (Figure 13.1). The kilning process also introduces flavor and colorproperties, which are very important in the subsequent beer production.

Malt is similar in appearance to barley grain, but closer physical exami-nation reveals that the embryo has developed considerably during maltingand that the endosperm has become friable (Figure 13.2). Substantialchanges have taken place in many of the constituents of the grain, specifi-cally in its cell walls, starch, and protein. During the malting process a con-siderable synthesis of enzymes takes place, and these enzymes are in turnresponsible for the major physical changes observed (Pollock, 1962).

The key process in beer production is the fermentation of the sugars con-tained in the wort to form alcohol and carbon dioxide (Figure 13.1) (Houghet al., 1987; Moll, 1991; Lewis and Young, 1995; Kunze, 1996). To provide

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FIGURE 13.1. Diagrammatic summary of malting and brewing processes, with the key events during these processesshown.

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the necessary conditions, initially insoluble components in the malt must berendered soluble by enzymes developed during germination; in particular,soluble fermentable sugars must be produced. The formation and dissolu-tion of these compounds is the purpose of wort production (Hough et al.,1987; Moll, 1991; Lewis and Young, 1995; Kunze, 1996). To accomplish it,malt is milled, mixed with water, and processed in one of two mash vessels,the mash tun or the mash kettle, to give as much soluble extract as possible.Where extra sources of starch (maize grits, rice, etc.) are added to the wort,they are processed, beforehand, in the adjunct cooker. In the lauter vessel(mash separation vessel), the soluble extract in the wort is separated fromthe insoluble material, called the spent grains. The wort is then boiled in thewort kettle with hops, which give beer its characteristically bitter taste. Thehot wort so produced is freed from the precipitated particles in a whirlpoolor centrifuge, subsequently cooled, and transferred to the fermentationtanks.

To transform wort into beer, the sugars in it must be fermented by en-zymes of the yeast to ethanol and carbon dioxide (Figure 13.1) (Hough et al.,1987; Moll, 1991; Lewis and Young, 1995; Kunze, 1996). Fermentationand conditioning (maturation) are carried out in fermentation and lager cel-lars at low temperature, to optimize yeast performance and to avoid the for-mation of undesirable by-products that could negatively affect both flavorand appearance of the final product. Under optimal conditions, the undesir-able by-products are either not formed or are removed. There are differentfermentation systems depending on the final beer desired (Hough et al.,1987; Moll, 1991; Lewis and Young, 1995); here yeast characteristics are ofparamount importance. The filtration process is intended to remove yeast

(a) (b)

FIGURE 13.2. High-power micrograph of a barley grain endosperm (a) and amodified malt endosperm (b) (Source: Courtesy of Sandy MacGregor.)

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and other turbidity-causing materials from beer (Moll, 1991; Kunze, 1996),and different filtration techniques are available (Kunze, 1996). The stabili-zation of beer, both microbiologically and colloidally, aims at keeping itsquality intact for as long as possible, thus preserving its shelf life undercommercial conditions. The microbiological stabilization uses differentsystems, e.g., pasteurization, flash pasteurization, cold sterile filling, and soon, whereas the colloidal stabilization makes use of different agents to pre-vent the formation of colloidal hazes (Hough et al., 1987; Hardwick, 1995;Kunze, 1996). Finally, beer is carbonated and filled in bottles or cansor kegsor is conditioned with additional yeast and filled in casks to be distributedfor sale.

Objectives

All the malting and brewing processes described act on the raw material,i.e., barley grains, the quality of which is strongly dependent on the compo-sition of these grains. This, in turn, is dependent on genotypic characteris-tics (conferred to them by breeding) and environmental effects (some ofwhich may be altered by crop management). In this chapter we review themajor grain structural components that affect malting quality in barley anddiscuss the genotypic and environmental factors that may modify it.

GRAIN STRUCTURAL COMPONENTSTHAT AFFECT MALTING QUALITY

Starch and Nonstarch Polysaccharides

Starch, protein, and -glucan comprise around 80 percent of barley grainweight (MacGregor and Fincher, 1993). It is the products of starch (by farthe main polysaccharide in grain cereals) hydrolysis that are fermented toalcohol in brewing. Therefore, genetic factors affecting the content ofstarch or the facility to hydrolyze it into simple sugars would be expected tohave consequences for malting and brewing quality.

Starch gives the main substrates for yeast to produce alcohol duringbrewing fermentation. In the mature barley grain it exists in granular form,with two distinct populations of large (A-type) and small (B-type) granules,the latter accounting for 90 percent of the total number but only 10 percentof the total volume (Bathgate and Palmer, 1972). Starch is a combination ofthe two polysaccharides amylose (AM) and amylopectin (AP), with APconstituting approximately 75 percent of the starch in normal, cultivatedbarley. In spite of the straight chain of AM and its molecular simplicity, AP

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is more readily degraded by amylases (Ellis, 1976). The high amylose char-acter is also associated with a reduction in the size of the A-type granules(Ellis, 1976), and the smaller the granules the more heavily they are imbed-ded into the surrounding protein matrix (Swanston, 1994). This gives rise toa very compact endosperm structure which is difficult to disrupt either en-zymatically or mechanically (Swanston, Ellis, and Stark, 1995). Thus, B-type granules are more difficult to degrade than the larger A-type granules,and stresses reducing the proportion of A-type granules tend to reduce maltextract, beyond any additional effect these stresses may have on the amountof starch. Allan and colleagues (1995) suggested that barley samples with agreater proportion of larger diameter starch granules could potentially pro-duce more fermentable sugars because they would have a greater percent-age of starch by volume. In addition, a portion of the AM forms a complexwith lipids which remains stable at temperatures above 90ºC (Tester andMorrison, 1993) and is not degraded during malting, this being exacerbatedin high AM genotypes (Swanston, Ellis, and Stark, 1995).

Nonstarch polysaccharides and lignin (~10 to 20 percent of grain) arestructural components of cell walls and negatively affect the water uptakeand germination of the barley grain (Molina-Cano, Swanston, and Ullrich,2000). Other fiber components that are partially soluble are mixed-linked(1 3, 1 4) -glucans (1.5 to 11.5 percent) and arabinoxylans (~4 to 8 per-cent), which have a tendency to increase the viscosity of solutions andcolloids, thus hampering the diffusion of water and enzymes throughout theendosperm. -glucans are the primary components in the starchy endo-sperm cell walls (Fincher, 1975). Arabinoxylans, also known as pentosans,are found primarily in the cell walls of the hull and aleurone (Han andFroseth, 1992).

High levels of -glucan have long been regarded as deleterious to malt-ing quality, as they may reduce the rate of endosperm modification (Bam-forth, 1982). Residual cell walls are barriers to amylolytic and proteolyticenzymes acting during malting and mashing. In addition, high levels of -glucan in the wort cause increases in viscosity, which may lead to filtrationproblems (Bamforth and Barclay, 1993). Barley -glucans are classed aswater soluble or insoluble and can be assessed separately by an enzymaticmethod (Aman and Graham, 1987).

Storage Proteins

The negative correlation between malt extract yield (i.e., the total ex-tractable material that is likely to be obtained from a given malt) and barleyprotein content, first reported by Bishop in the 1930s (Molina-Cano, Swan-

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ston, and Ullrich, 2000), is now known to be mainly due to the hordeins, themajor fraction of the endosperm storage proteins in barley grains. There aretwo main reasons for this negative correlation: (1) the relative increase inother grain components naturally implies a likely reduction in starch con-tent, the main source of extract (Smith, 1990), and (2) hordein acts as aphysical barrier to starch degradation due to its role as the main componentof the endosperm protein matrix into which the starch granules are embed-ded. Increases in hordein content thus imply a restricted access to the amy-lolytic enzymes during malting (Palmer, 1989).

When examining cultivar and environmental effects on malting qualityof Australian barleys grown under a Mediterranean-type climate (Eagleset al., 1995), it was found that seasonal differences in malt extract levelscould not be explained completely by differences in protein concentration.This indicates that other factors, such as the type of protein present or thestarch characteristics, influence malt extract independently of grain proteinconcentration.

Hordeins are soluble in alcohol/water mixtures and, on average, accountfor up to 60 percent of the total grain nitrogen (Brennan et al., 1998). Theyare classified into four groups, or families of polypeptides, called B-, C-, D-,and -hordeins, and are encoded by the genes Hor2 (B-fraction), Hor1(C-fraction), Hor3 (D-fraction), and Hor5 ( -fraction), located on barleychromosome 5(1H) (Kreis and Shewry, 1992). The B- and C-fractions ac-count for 70 to 80 percent and 10 to 20 percent, respectively, of the totalhordein, while the D and groups are quantitatively minor components.

There is still uncertainty on the specific effect of the different hordeinsubunits on malting performance (Swanston and Ellis, 2002). Furthermore,the results may differ among the various methodologies used to measure thehordein fractions (Janes and Skerritt, 1993) and/or a clear definition of thedifferent groups that conformed a certain fraction (reviewed by Smith,1990). For example, some researchers found a negative relationship be-tween B-hordein (Baxter and Wainwright, 1979), D-hordein (Howard et al.,1996) or a colloidal aggregate of D- and B-hordeins linked by thiol groups,named gel protein (van den Berg et al., 1981; Smith and Lister, 1983), andmalting quality. Recently, analyses of isogenic barley lines with and with-out D-hordeins clearly demonstrated that D-hordein was a major compo-nent of gel protein but failed to observe differences in malting performance,raising uncertainties about the deleterious influence of this hordein fractionon malting quality. As a continuation of this work, the same six pairs ofnear-isogenic lines, differing in the presence or absence of D-hordein, wereincluded in genetic backgrounds with different B- and C-hordein alleles(Brennan et al., 1998). The conclusion reached by the authors was that dif-

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ferences in malting quality were not related to either the presence or ab-sence of D-hordein or to gel protein levels.

Additional effects of hordein subunits on other malting attributes werefound. Peltonen and colleagues (1994) evaluated the effect of D-, C-, and B-hordeins on the malting quality of northern European barleys. They foundthat the B-fraction had some effect on malting quality by regulating dia-static power (a complex of enzymes that hydrolyze starch in barley endo-sperms). In the absence of a reducing agent in the extraction solvent, a highconcentration of B-hordein was correlated with an increase in extract yield.When environmental conditions favored high nitrogen uptake efficiency, alarger proportion of D-hordein disulphide bonds was synthesized, thus de-creasing malting quality. It was speculated that with lower grain nitrogencontent and lower D:B hordein ratio, malting quality increased (Peltonenet al., 1994). Contradictory results were obtained, however, by Molina-Cano, Swanston, and Ullrich (2000), who found that B-hordeins were asso-ciated with a decrease of malt extract, which was significantly greater inNordic compared to Mediterranean barleys.

Study of the malting behavior of barleys grown in Spain and Scotland(Molina-Cano et al., 1995) permitted the formulation of a model stating thata lower content of insoluble -glucans and a higher B/C-hordein ratio waslinked to superior malting quality. The use of total grain protein content as apredictor of malting quality was considered to be inadequate to fully ac-count for the different malting behavior of northern and southern Europeanbarleys (as also discussed in Savin and Molina-Cano, 2002).

Further research into genetic (G) and environmental (E) influences onhordein content was carried out by Molina-Cano and colleagues (Molina-Cano, Polo, Romera, et al., 2001; Molina-Cano, Polo, Sopena, et al., 2001),using induced mutants in the malting barley cultivar Triumph that were grownin both Spain and Scotland. The mutant TL 43 showed higher B-hordeincontent than ‘Triumph’ in Scotland but lower content in Spain; it displayedconsistently higher C- and D-hordein content than ‘Triumph’ in both envi-ronments, i.e., there was GxE interaction for B-hordein and none for C- andD-hordein content. There were also differences in grain ultra-structure be-tween the two lines, as TL43 showed a more dense protein matrix than ‘Tri-umph,’ together with a thinner pericarp, testa, and aleurone layers (Molina-Cano, Polo, Sopena, et al., 2001). When studying water uptake, a generalconclusion was that although influenced both by genotype and environ-ment, water uptake was more dependent on the latter. B-hordein quantityand distribution were important factors hampering water uptake, but solu-ble -glucan content, previously implicated in determining differences inwater uptake between grains produced in Spain and Scotland, appeared tohave little effect in this study (Molina-Cano, Polo, Sopena, et al., 2001).

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Endosperm modification during malting in the same genotypes as, i.e.,TL43/‘Triumph,’ was also studied in Spain and Scotland during differentseasons (Swanston and Molina-Cano, submitted). Over seasons, the mutantgenerally gave slightly lower extracts than ‘Triumph,’ but this was associ-ated with higher protein levels in the malted grain. At given nitrogen levels,TL43 had a similar extract to ‘Triumph,’ but a higher Kolbach index, sug-gesting that the extract contained higher proportions of protein-derived ma-terial. Patterns of development of both extract and fermentability, betweentwo and five days after the initiation of germination, were determinedmainly by the genotype, but the environment influenced the levels obtainedfor both traits. Two days after the initiation of germination, TL43 grown atLleida, Spain, gave higher soluble nitrogen than ‘Triumph’ but, thereafter,the relative rates of nitrogen solubilization were similar for both cultivars.At both experimental sites (Scotland and Spain), TL43 gave extracts with ahigher proportion of nitrogenous material than ‘Triumph’ and, conse-quently, fermentability was always lower in the mutant.

GENOTYPIC AND ENVIRONMENTAL FACTORSAFFECTING MALTING QUALITY

The environment during grain filling may produce important changes ingrain composition and quality in cereals (see Chapter 11; Gooding andDavies, 1997; Savin and Molina-Cano, 2002). However, genotypic differ-ences in the response to the environment can also be important (Stone andNicolas, 1994; Savin and Nicolas, 1996; Wallwork et al., 1998b). More-over, genotype environment interaction is one of the causes of unpredict-able variation found in quantitative traits such as malting quality (Molina-Cano, Polo, Romera, et al., 2001). In this section, the major environmentalfactors involved in the determination of malting quality are discussed, con-sidering also genetic variability in the response to those factors.

High Temperatures

It is well known that temperature can have a profound impact on crop de-velopment and grain yield (e.g., Slafer and Rawson, 1994). For example, itis well established that optimum temperature for maximum grain weight intemperate cereals is around 15 to 18°C (e.g., Chowdhury and Wardlaw,1978). However, in most barley-growing areas, mean temperature duringgrain filling is higher than this optimum.

High temperatures can also have an important effect on grain composi-tion and quality. The responses to high temperatures have been divided into

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two categories (Stone and Nicolas, 1994, Wardlaw and Wrigley, 1994):(1) those resulting from sustained periods of moderately high temperature(25 to 30 to 32°C) and (2) those originated from brief periods (three to fivedays) of very high temperature (ca. >35°C). This division presumes that thetype of responses and the mechanisms involved differ between these twothermal ranges (Wardlaw and Wrigley, 1994). Plant responses to moder-ately high temperature result largely from changes in the rate and durationof existing processes. In contrast, under very high temperatures some pro-cesses are severely retarded while other physiological processes may be in-duced or intensified.

Responses to Moderately High Temperature

The temperature range from 15 to 32°C involves a progressive decreasein grain size with temperature increase (Chowdhury and Wardlaw, 1978;Wardlaw and Wrigley, 1994). Although grain growth rate increases withtemperature, the duration of grain growth is reduced to a greater extent(e.g., Chowdhury and Wardlaw, 1978). In this range, wheat yield has beenshown to decline approximately 3 to 4 percent for each 1°C rise in averagetemperature above 15°C (Wardlaw and Wrigley, 1994) under both con-trolled and field conditions. Barley shows the same trend but with an appar-ently reduced sensitivity. For instance, Savin and colleagues (1997a) showeda reduction in grain weight with increases in temperature of only 1 per-cent/°C (Figure 13.3a).

It is commonly accepted that accumulation of starch is more sensitive tohigh temperature than protein accumulation (Jenner, Ugalde, and Aspinall,1991), resulting in a reduction in relative starch content (Figure 13.3a). Insome experiments, there were reductions in the number of both A- andB-type starch granules due to heat stress (MacLeod and Duffus, 1988).However, Savin and colleagues (1997a) showed that moderately high tem-peratures (27 or 30°C) commencing 20 days after anthesis and lasting untilmaturity reduced the final size of the grains and their starch content with noclear trends in starch granule number.

In general, grain nitrogen content per grain was unchanged by moder-ately high temperatures (Figure 13.3a), but grain nitrogen percentage increased(Glennie-Holmes and Jacobsen, 1994; Savin et al., 1997a). In addition, pro-tein composition may change under elevated temperature. Swanston andcolleagues (1997) found changes in the B:C-hordein ratio when comparingresults obtained in Scotland and Spain with the same cultivar. B:C-hordeinwas lower in the hotter environment of Spain.

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Differences between sites in temperature during grain filling may alsochange -glucan deposition. Swanston and colleagues (1997) found differ-ences in the pattern of -glucan deposition during grain filling between bar-ley crops grown in Spain and Scotland, with Spanish-grown samples havinghigher levels of total -glucan but lower levels of the insoluble fraction. In astudy aimed to characterize the differences between the malting behavior ofScandinavian and Iberian barleys, Molina-Cano and colleagues (2002)pointed out that total and insoluble -glucans are of paramount importance.They were an effective barrier to extract development in the North, but wereassociated with an increase in the South. A conclusion was that -glucans inthe Iberian barleys contributed to extract as a consequence of the capacityof these barleys to synthesize and release -glucan hydrolazes during ger-mination.

A general consequence of exposure to moderately high temperaturesduring grain filling under controlled conditions was a reduction in malt ex-tract (Figure 13.3b) (Glennie-Holmes and Jacobsen, 1994). The effect isevident, even after exposure for only a few days to moderately high temper-atures. For instance, when the spikes (but not the whole canopy) were ex-posed to brief periods (five days) of high temperatures (Passarella, Savin,

FIGURE 13.3. Theoretical reduction of grain weight, starch, and protein absolutecontent (a) and starch and protein relative content (b) as temperature increasesfrom an initial temperature (T0) during grain filling. (Source: References for thesedata can be found in Jenner, Ugalde, and Aspinall, 1991; Savin et al., 1997a;Savin and Molina-Cano, 2002 [a]; and Glennie-Holmes and Jacobsen, 1994;Savin and Molina-Cano, 2002; Passarella, Savin, and Slafer, 2002 [b].)

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and Slafer, 2002), malt extract was reduced by ca. 2 percent. This figure, al-though apparently small, is rather important considering the very slight en-vironmental change (the spike temperature was about 30ºC at midday foronly five days) and there was also a reduction in the amount of maltablegrains (as the proportion of grains <2.5 mm increased).

Responses to Brief Periods of Very High Temperature

Brief periods of very high temperature are quite common during thegrain-filling phase of cereal crops in temperate areas (MacNicol et al.,1993; Stone and Nicolas 1994). Although these short episodes of high tem-perature do not greatly change the average temperature during the wholegrain-filling period, they can affect both grain yield and quality in wheat(Stone and Nicolas, 1994) and barley (Savin and Nicolas, 1996; Savin,Stone, and Nicolas, 1996). This type of stress reduces grain weight by 5 to30 percent depending on the cultivar, time of exposure, and duration of thestress (Savin and Molina-Cano, 2002).

Reductions in grain weight are, in general, closely matched with starchcontent per grain (Savin and Molina-Cano, 2002) and are more related tothe reduction in the number of B-type than A-type starch granules (Savinand Molina-Cano, 2002). This reduction may be caused by an irreversibleeffect of heat stress on the activity of soluble starch synthase, a key enzymefor the synthesis of starch (Wallwork et al., 1998a).

Similar to the effects of continuous moderately high temperatures, grainprotein percentage is frequently increased when grains are exposed to briefperiods of very high temperature. The increase in protein percentage is pri-marily due to a reduction in grain starch content, as nitrogen accumulationis comparatively unresponsive to brief episodes of very high temperatures(Savin et al., 1997b; Savin and Nicolas, 1996, 1999; Wallwork et al., 1998a).

-glucan accumulation in the grains begins approximately 15 days afteranthesis (Aman, Graham, and Tilly, 1989). Heat stress early in the accumu-lation phase could reduce -glucan content more severely than a later stress(Savin et al., 1997b). For example, when heat stress commenced 15 days af-ter anthesis, -glucan content was significantly reduced in heat-stressedplants (Savin et al., 1997b; Wallwork et al., 1998b), whereas when the stresscommenced later it was not significantly affected (Savin and Nicolas, 1996;Savin, Stone, and Nicolas, 1996).

Exposure to brief periods of very high temperature reduced hot water ex-tract in some experiments (Wallwork et al., 1998b; Savin, Stone, andNicolas, 1996; Savin et al., 1997b) but not in others (MacNicol et al., 1993;Savin et al., 1997b). This could be an indication that individual components

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such as starch, nitrogen, or -glucan content cannot individually explainmalt extract values when barley plants are exposed to short periods of hightemperature. It is likely that interactions among these quality attributes maybe responsible for the limited effect of those stresses on malt extract in mostexperiments (Savin and Molina-Cano, 2002).

Drought

In most of the rainfall regions of the world, small cereal grains are sub-jected to water stress which may occur at different stages during the lifecycle. Traditionally, barley is grown in places with less availability of re-sources than wheat. However, barley is usually the highest-yielding temper-ate cereal in low rainfall areas where there is a Mediterranean-type climate(López-Castañeda and Richards, 1995). The yield advantage of barley isparticularly evident under dry conditions, but it may disappear or be re-versed when water is not a limiting factor (Araus, Slafer, and Romagosa,1999). Little is known about the effects of postanthesis drought on grainquality in cereals, as only a few experiments have been performed to assessthese effects. In addition, a problem with many drought experiments is thatthe intensity and timing of the stress, relative to grain growth, are oftenpoorly defined, so that results are difficult to interpret or compare (Savinand Molina-Cano, 2002).

Grain filling depends partly on current photosynthesis and partly on thetransfer of assimilate accumulated before flowering (Bonnett and Incoll,1992). The amount of assimilate coming from photosynthesis after flower-ing depends on the efficiency with which the plants can use the limited wa-ter available during grain filling (Passioura, 1994). On the other hand, wa-ter-limited plants may translocate considerable amounts of preanthesisassimilates to the grain. The proportion of grain weight that comes from thissource varies widely with species and environments and depends stronglyon the pattern of drought (Passioura, 1994).

Only five reported studies appear to have examined the effects of droughton malting quality (reviewed in Savin and Molina-Cano, 2002). Experi-ments have been conducted in greenhouses (Savin and Nicolas, 1996,1999), growth chambers (Morgan and Riggs, 1981; MacNicol et al., 1993),and under field conditions (Coles, Jamieson, and Haslemore, 1991). Grainweight reduction varied between 3 and 30 percent compared to the well-watered control, depending on the intensity and timing of exposure as wellas the genotype (Savin and Molina-Cano, 2002). This grain weight reduc-tion seems to have occurred primarily because starch accumulation hadbeen reduced by drought. Apparently, the final number of the endosperm

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cells was not altered by water stress (Brooks, Jenner, and Aspinall, 1982),but the size or number of the A-type or B-type starch granules in the endo-sperm decreased, depending on the timing of the water stress (Brooks, Jen-ner, and Aspinall, 1982; Savin and Nicolas, 1999).

Morgan and Riggs (1981) found that barley extract viscosity (an indica-tor of -glucan content) increased with drought applied from anthesis on-ward. However, when severe (Savin and Nicolas, 1996) or mild (Savin andNicolas, 1999) droughts were applied, there was a tendency for -glucancontent in the grains to be reduced. This is in agreement with results re-ported under field (Stuart, Loi, and Fincher, 1988; Coles, Jamieson, andHaslemore, 1991) and growth chamber conditions (MacNicol et al., 1993).Savin and Molina-Cano (2002), reviewing results from several drought ex-periments, showed a linear relationship between malt extract and -glucandegradation (i.e., difference between the percentage of -glucans in grainand in malt, when the latter is given as a percentage, Stuart, Loi, andFincher, 1998).

Nutrient Availability

Nitrogen is the nutrient that most often limits crop yield. Nitrogen playsnumerous key roles in plant biochemistry and is an essential constituent ofenzymes, chlorophyll, nucleic acids and storage proteins. Consequently, adeficiency in the supply or availability of nitrogen may have a significantinfluence on crop yield and grain quality. In malting barley, nutrient man-agement is essential because high nitrogen availability may increase yieldbut could also be detrimental to quality, in contrast to the situation in bread-making wheat (see Chapter 11). As discussed earlier, a high grain nitrogencontent is inversely related to malting quality (Smith, 1990; Howard et al.,1996). Therefore, the amount of soil nitrogen required to maximize yieldand quality would differ for each particular combination of genotype andenvironment.

However, grain yield and grain protein concentration are generally nega-tively correlated in most of the cropping systems (Smith et al., 1999; Stoneand Savin, 1999). The amount of carbohydrates accumulated in a smallgrain cereal is most often sink limited during grain filling, whereas theamount of nitrogen is usually source limited under normal field conditions(Dreccer, Grashoff, and Rabbinge, 1997). The final protein concentrationwill thus depend on the nitrogen availability during the crop cycle (Stoneand Savin, 1999). For instance, several authors reported an increase ofC-hordeins under conditions of high fertility (Shewry et al., 1983).

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ACHIEVING BARLEY-GRAIN QUALITY TARGETS

Breeding

The Analytical Assessment of Malting Qualityin Breeding Programs

The analytical methods used to evaluate malting quality in samples frombreeding programs must be tailored for such purposes. The high number ofsamples to be analyzed, their small size, and the short time available to pro-duce the results (Molina-Cano, 1991) greatly condition the methodology,which should also provide the lowest possible operating costs. As yet, suffi-ciently rapid methods have not been designed, since malting is essential,nor are the available ones cheap enough (the average cost of a fully ana-lyzed malt sample is between 50 to 150 U.S. dollars, depending on the num-ber of analytical parameters measured). Furthermore, the greater variabilityof nitrogen content between grains derived from ear-to-row progenies, thanin grains harvested from dense plots, makes the quality data obtained duringthe visual selection phase of the breeding program (F2 to F5) rather unreli-able. Other complicating factors during this phase of the program are GxEinteraction and heterozygosity.

The predictive methods developed in the first half of the twentieth cen-tury, analyzing unmalted grain, by the Bendelow and Meredith team (re-viewed in Bendelow, 1981) determined the potential extract and the amylo-lytic activity in previously digested barley flour, in the first case with asolution of malt enzymes and in the second with papain. Finally, it was de-termined that malting is essential if one wants an accurate quality assess-ment (Bendelow, 1981, personal communication).

Other nonmalting methods include milling energy (Allison et al., 1979),the measure of the viscosity of an acid extract of barley flour, as a predictorof -glucan content (Aastrup, 1979), sedimentation (Reeves et al., 1979),falling time (Morgan, 1977), and barley hordein content (Baxter and Wain-wright, 1979). Though useful under certain conditions, none of these meth-ods is widely applicable (Bendelow, 1981).

The development of enzymatic (McCleary and Glennie-Holmes, 1985),high performance liquid chromatography (HPLC) (Pérez-Vendrell et al.,1995), and fluorimetric (Jørgensen and Aastrup, 1988) methods has en-abled breeders to readily select for low levels of -glucan in barley grain.

The first micromalting devices were developed in the 1950s, such asthose of the Plant Breeding Institute in Cambridge, United Kingdom (Whit-more and Sparrow, 1957), or the Canadian Grain Research Laboratory (At-

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kinson and Bendelow, 1976). They had capacity for a reasonable number ofvery small samples, but they were manually operated and thus expensiveand cumbersome, besides having a limited level of precision. Completelyautomated and computer-controlled micromalting models have now beendeveloped, with an enlarged capacity of 100 to 300 samples, depending onsample size (Gothard, Morgan, and Smith, 1980; Takeda et al., 1981; Glen-nie-Holmes, Moon, and Cornish, 1990). Other devices include those ofCarlsberg and Abed of Denmark and Phoenix and Joe White of Australia.An interesting development was the micromalting equipment of the Scot-tish Crop Research Institute, which allows assessment of experimental datalaid out with complex statistical designs (Swanston, 1997).

Although evaluation of a commercial malt can be based on a large num-ber of parameters (European Brewery Convention [EBC], 1987), the as-sessment of breeding samples relies on a smaller number of quality parame-ters. They can be summarized in a single figure or index, such as the EBCquality index Q (Molina-Cano, 1987) or the Carlsberg index (Ingversen,Englyst, and Jorgensen, 1989), thus supplying the breeder with a singlescore with likely predictive value for quality selection.

Breeding Barley for Malting in the Molecular Era

The classical barley breeding programs were conceived and carried outwith the aid of the methods described previously which have been reliableand widely accepted over the years. New tools offered by DNA methodolo-gies will have an increasing role in present and future malting barley breed-ing efforts, mainly because with these methods it is possible to directly ob-serve the genotype, rather than the phenotype, in contrast to the classicalmethods used. As Swanston and Ellis (2002) pointed out, barley breedersare now able to exploit the knowledge that has been gained on factors af-fecting quality and to use DNA markers, such as restriction fragment lengthpolymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs),and amplified fragment length polymorphisms (AFLPs), to identify the ge-netic basis of desired traits to be selected in breeding.

The development of different genetic maps locating quantitative traitloci (QTLs) affecting quality traits has brought about a wealth of knowl-edge to aid malting barley breeders. Updated reviews on this topic includeKleinhofs (2000), Hayes and Jones (2000), Meyer and colleagues (2000),Molina-Cano, Swanston, and Ullrich (2000), Barr and colleagues (2000),Swanston and Ellis (2002), and Ullrich (2002). The rapid development ex-perienced in this field should oblige both breeders and geneticists to update

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their knowledge through the World Wide Web. The main sites that we areaware of at the time of writing this chapter include the following:

• Washington State University Barley Genomicshttp://barleygenomics.wsu.edu

• Institute of Plant Genetics and Crop Plant Research—Gaterslebenhttp://www.ipk-gatersleben.de/en/

• Barley Genetics Newsletterhttp://wheat.pw.usda.gov/ggpages/bgn

• U.S. Department of Agriculture GrainGenes Databasehttp://wheat.pw.usda.gov/ggpages/outline.html

• Molecular Breeding (journal)http://www.kluweronline.com/issn/1380-3743

• North American Barley Genome Projecthttp://barleyworld.org

• Scottish Crop Research Institutehttp://www.scri.sari.ac.uk/

Barley breeders have made significant changes and improvements in thecrop, and today the malting industry has access to high quality raw material.This has been achieved largely as a result of phenotypic selection (Swan-ston and Ellis, 2002), making use of increasingly sophisticated testing pro-cedures. Malting, however, comprises a highly complex series of interre-lated biochemical pathways occurring simultaneously, so the underlyinggenetic control is equally complex. In recent years, major steps have beentaken toward locating some of the critical genes and assessing their role.This is likely to continue, making use of techniques such as expressed se-quence tags (ESTs), where the messenger RNA associated with activatedgenes is extracted to clone sequences of cDNA. One possible application ofESTs to malting barley has already been outlined briefly (Swanston et al.,1999). The vast improvements in information technology of recent yearsalso make it possible to compare cDNA sequences with those alreadyknown and held in databases; around 70 percent of the ESTs from maltedbarley showed homology with known sequences (Swanston et al., 1999).Not all sequences that have been determined have a known function, but ithas been possible to detect sequences associated with both carbohydrateand amino acid metabolism. Future developments are likely to includemapping of ESTs and comparison of map locations with those of knowngenes or QTLs.

The future, beyond any doubt, will arise from the interaction of classicalbreeding, genomics, and proteomics, although genetic engineering will

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also have an important role. Updated accounts on these topics were recentlyoffered by Kleinhofs (2000), Waugh (2000), Lörz and colleagues (2000)and Fincher (2000).

Crop Management

Although both grain yield and quality are determined throughout thegrowing season, important decisions that will strongly affect them shouldbe taken before sowing (Calderini and Dreccer, 2002). Among others, thechoice of the genotype and the amount of nitrogen available are central forsuccessfully combining the genotype potential for yield and quality withthe environmental availability of resources. Final grain quality is the resultof the interaction between the genotype, the natural environment, and cropmanagement practices (Gooding and Davies, 1997). In extensive produc-tion systems it is not possible to provide each stage of the crop cycle withthe optimal combination of environmental factors to reach the highest pos-sible yield and quality; therefore, a trade-off is to make presowing decisionsto ensure that critical crop stages for the definition of yield and quality aregiven a preferential environment (Calderini and Dreccer, 2002). Neverthe-less, knowledge of the effects of environment and GxE interaction is stillrather imprecise, so management strategies with the objective of increasingyields, while obtaining high malting quality barley, are difficult to design.In this section only a brief discussion on how crop management may mod-ify barley malting quality is presented.

Grain yield is more strongly related to grain number than to grain size,with individual grain weight being the most stable component of yield(Smith et al., 1999). Thus, in general, grain yield is more responsive to thepreanthesis period conditions than to those occurring after anthesis. How-ever, grain quality may be determined by responses during both the pre- andpostanthesis periods. A strong relationship can occur between plant proteincontent at anthesis (which is the result of crop nitrogen absorption beforeanthesis) and final grain protein content (Molina-Cano, Gracia, and Ciudad,2001). The synthesis of the different grain components and the final graincomposition can be altered by high temperatures, drought, and nitrogenavailability during grain filling as discussed previously (MacNicol et al.,1993; Savin and Nicolas, 1996, 1999; Wallwork et al., 1998a,b) in relationto the length of the grain growth period as an environmental factor (Savinand Molina-Cano, 2002). Therefore, choosing both the appropriate cultivarfor the environment and the amount of nitrogen fertilizer to apply are keyaspects of crop management.

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Choosing Cultivars

Malting barley is a crop sold on the basis of cultivar; therefore, cultivarchoice strongly determines whether the grains will be accepted by the in-dustry. Moreover, it is a common situation that contracts with the maltingcompanies include the obligation of sowing certain cultivars, although ac-ceptance of the grain is subjected to several additional requirements set bythe company.

Genotypes are generally grouped into different classes accordingto (1) time of sowing: winter, Mediterranean, and spring cultivars and/or(2) spike type: two- and six-row cultivars. The use of a particular group willbe associated with climatic considerations, mainly the length of the grow-ing season and the likelihood of freezing temperatures during winter (Cal-derini and Dreccer, 2002). The first step toward choosing the best genotypefrom the broad range of possibilities defined by the combination of thesetwo groupings (winter-spring barley or two-six rowed barley) is to clearlyand precisely define the agroclimatic characteristics of the crop productionsystem (Calderini and Dreccer, 2002).

Nitrogen Fertilizer

Addition of nitrogen fertilizer is one of the most frequently used meth-ods for altering grain yield and quality (Stone and Savin, 1999). Startingfrom a low level of nitrogen availability, the first increment of nitrogen fer-tilizer increases the amounts of both starch and protein in the grain, but theresponse of starch is usually the greater (Figure 13.4).

Therefore, it tends to increase yield but decrease protein percentage, re-sulting in the frequently reported negative relationship between grain yieldand protein percentage (see Stone and Savin, 1999). Before the critical levelof nitrogen is attained, the response of starch and protein accumulation en-ters a second region of response (Figure 13.4), in which additional nitrogenfertilizer will often have a reduced (but still positive) effect on starch accu-mulation and a proportionally greater impact on protein accumulation. Thenet effect of nitrogen in the second region of response is therefore a smallincrease in yield and a comparatively large increase in protein percentage(Figure 13.4). As greater amounts of nitrogen are added, the crop may reachthe third region of response (Figure 13.4), at which maximum yield hasbeen attained. In this region, additional fertilizer does not affect the amountof starch in the grain, but it does increase the amount of grain protein. As aresult, protein percentage is highly responsive to nitrogen in this “luxuryconsumption” region of nitrogen addition. Thus, addition of nitrogen to a

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soil that is rather poor in terms of nitrogen availability increases yield,whereas fertilizing a crop when the soil has a relatively high nitrogen levelincreases protein percentage (Stone and Savin, 1999). In barley crops formalting purposes the decision to add nitrogen fertilizer is more critical thanin wheat crops for bread making, since the objective is to have high grainyields, so sufficient nitrogen must be present to achieve this; on the otherhand, nitrogen should not lead to grain protein levels high enough to cause anegative relationship between malt extract and protein content (Molina-Cano, Polo, Romera, et al., 2001). Therefore, the final decision on theamount of nitrogen fertilizer to add should come from the expected yield re-sponses at each site and also depend on the temperature, water availability,and the type of malt required by the local industry.

CONCLUSIONS

Malting quality is more than the sum of the contributions of starch, pro-tein, and -glucans to malt extract or any other measure of quality. It is notjust the presence of a given constituent that determines quality, but ratherthe interaction between different constituents. In addition, the final compo-sition of the barley grains may be modified by environment and crop man-agement. Understanding the influence of the major environmental factorson grain composition and how these changes affect malting performance isa major factor in obtaining the quality required.

FIGURE 13.4. Diagrammatic representation of the response of yield (—) andprotein percentage (—–) to nitrogen fertilizer (Source: From P. J. Stone andR. Savin, 1999, Grain quality and its physiological determinants. In E. H. Satorreand G. A. Slafer (Eds.), Wheat: Ecology and Physiology of Yield Determination,pp. 85-120. Reproduced with permission of The Haworth Press, Inc.)

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In recent years important improvements have been achieved in tech-niques for measuring the different components of grain and malt and also inthe selection of high quality raw material through different breeding tech-niques. This is likely to continue in the future. However, a better under-standing of how particular environmental factors may modify the composi-tion of the barley grain and its subsequent transformation into malt and beeris still lacking. A better understanding of how these factors interact is alsorequired to predict the final quality of the barley crop under different cropmanagement regimes and environmental conditions.

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Stone, P.J. and Nicolas, M.E. (1994). The effects of short periods of high tempera-ture during grain filling on grain yield and quality vary widely between wheatcultivars. Australian Journal of Plant Physiology 21: 887-900.

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Stone, P.J. and Savin, R. (1999). Grain quality and its physiological determinants.In Satorre, E.H. and Slafer, G.A. (Eds.), Wheat: Ecology and Physiology of YieldDetermination (pp. 85-120). Binghamton, NY: Food Products Press.

Stuart, I.M., Loi, L., and Fincher, G.B. (1988). Varietal and environmental varia-tions in (1-3, 1-4)- -glucan levels and (1-3, 1-4)- -glucanase potential in barley:Relationships to malting quality. Journal of Cereal Science 7: 61-71.

Swanston, J.S. (1994). Malting performance of barleys with altered starch composi-tion. PhD thesis. Edinburgh, Heriot-Watt University.

Swanston, J.S. (1997). Assessment of malt. Ferment 10: 29-34.Swanston, J.S. and Ellis, R.P. (2002). Genetics and breeding of malt quality attrib-

utes. In Slafer, G.A., Molina-Cano, J.L., Savin, R., Araus, J.L., and Romagosa, I.(Eds.), Barley Science: Recent Advances from Molecular Biology to Agronomyof Yield and Quality (pp. 85-114). Binghamton, NY: Food Products Press.

Swanston, J.S., Ellis, R.P., Pérez-Vendrell, A., Voltas, J., and Molina-Cano, J.L.(1997). Patterns of barley grain development in Spain and Scotland and their im-plication for malting quality. Cereal Chemistry 74: 456-461.

Swanston, J.S., Ellis, R.P., and Stark, J.R. (1995). Effects on grain and malting qual-ity of genes altering barley starch composition. Journal of Cereal Science 22:265-273.

Swanston, J.S. and Molina-Cano, J.L. The relationship between protein solubil-isation and malting quality in a mutant of cv. Triumph. Journal of the Institute ofBrewing (submitted).

Swanston, J.S., Thomas, W.T.D., Powell, W., Meyer, R., Machray, G.C., and Hed-ley, P.E. (1999). Breeding barley for malt whisky distilling—A genome basedapproach. In Campbell, I. (Ed.), Proceedings of the Fifth Aviemore Conferenceon Malting, Brewing and Distilling. London: Institute of Brewing.

Takeda, G., Sekiguchi, T., Kurai, K., and Seko, H. (1981). New procedure for mi-cro-malting and its application for quality selection in barley breeding. JapaneseJournal of Breeding 31: 414-422.

Tester, R.F. and Morrison, W.R. (1993). Swelling and gelatinization of cerealstarches: VI. Starches from waxy Hector and Hector barleys at four stages ofgrain development. Journal of Cereal Science 17: 11-18.

Ullrich, S.E. (2002). Genetics and breeding of barley feed quality attributes. InSlafer, G.A., Molina-Cano, J.L., Savin, R., Araus, J.L., and Romagosa, I. (Eds.),Barley Science: Recent Advances from Molecular Biology to Agronomy of Yieldand Quality (pp. 115-142). Binghamton, NY: Food Products Press.

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Wallwork, M.A.B., Logue, S.J., MacLeod, L.C., and Jenner, C.F. (1998a). Effect ofhigh temperature during grain-filling on starch synthesis in developing barleygrain. Australian Journal of Plant Physiology 25: 173-181.

Wallwork, M.A.B., Logue, S.J., MacLeod, L.C., and Jenner, C.F. (1998b). Effectsof a period of high temperature during grain filling on the grain growth charac-

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teristics and malting quality of three australian malting barleys. Australian Jour-nal of Agricultural Research 49: 1287-1296.

Wardlaw, I.F. and Wrigley, C.W. (1994). Heat tolerance in temperate cereals: Anoverview. Australian Journal of Plant Physiology 21: 695-703.

Waugh, R. (2000). Current perspectives in barley genomics. In Logue, S. (Ed.),Barley Genetics VIII: Proceedings of the Eighth International Barley GeneticsSymposium, Volume I, Invited Papers (pp. 205-211). Glen Ormond: AdelaideUniversity.

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Wrigley, C.W. (1994). Developing better strategies to improve grain quality forwheat. Australian Journal of Agricultural Research 45: 1-17.

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Index

Page numbers followed by the letter “f” indicate figures; those followed by the letter“t” indicate tables.

abscisic acid (ABA)and dehydration, 153and dormancy, 171-175, 178-181,

186, 187, 190, 205release from, 235

and embryo growth, 224, 236and endosperm, 228, 233and genetics, 235and imbibition, 235and light, 224, 235and recalcitrance, 324, 334role of, 107, 131, 153, 235

Acer spp., 308, 313acetaldehyde, 149acorns, 307-308, 320adenosine diphosphate (ADP), 150adenosine triphosphate (ATP), 113, 150adventitious roots, 104-105aeration, 7-8, 11, 18-20, 135Aesculus hippocastanum

cryopreservation, 332desiccation sensitivity, 310, 312and temperature, 257-259, 309

after-ripeningand base water potential, 63, 257and dormancy, 180and soil water content, 264and sprouting samples, 209and temperature, 181, 256-259

aggregate size, 21, 23aging. See also deterioration

in leeks, 293oxidative damage, 154, 282-288,

290and priming, 142, 293, 294recalcitrance versus orthodoxy,

273-275, 309slow versus accelerated, 284

agriculture. See also specific cropsprediction, 78-82, 112success factors, 4, 8

agrochemicals, 129, 447. See alsofungicides

albumin, 363aleurone, 202, 205, 363alfalfa, 112, 129almond seeds, 284alpha-linolenic acid, 399-400alpha-tocopherol. See tocopherolamino acids

and deterioration, 280digestibility, 409essential, 408in oats, 362oilmeal, 407-408and oxidative damage, 285in rice, 363in wheat, 373-374

amphipathic substances, 315amplified fragment length

polymorphism (AFLPs), 183,444

amylasesand maturity, 202measuring, 211-212and priming, 294in rice, 264and sprouting, 204in wheat, 206, 207

amylograph, 211amylopectin, 380, 433amylose, 366, 367, 380, 433animal feed

barley and oats, 360-361harm from, 411-412oilmeals, 406phytic acid, 409

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animal feed (continued)and preharvest sprouting, 200suitability factors, 355

anoxia, 7, 110, 325. See also hypoxiaanticarcinogenicity, 406, 409antioxidants/free radical scavengers

and (de)(re)hydration, 314, 316phytic acid, 409and priming, 294role, 153in vegetable oils, 401-406

Aporusa lindleyana, 324aquatic plants, 7, 319Arabidopsis spp.

embryo expansion, 233and ethylene, 235genetics, 147, 157gibberellens, 233, 234and light, 223, 233, 263precocious germination, 171protein reserves, 150storage longevity, 291-292and tocopherol, 403

arabinosidase, 147, 227-228arabinoxylans, 434Araucaria spp., 307, 308, 327archaeology, 372arid conditions

and barley, 441drought tolerance, 114and myxospermy, 103-104seedbed preparation, 21soil compaction, 4, 8

artificial seeds, 334-335Artocarpus altilis, 318ascorbic acid, 290, 291assimilates, 441atherosclerosis, 396-398autoxidation, 282-285, 295, 398Avena fatua, 186, 187Avicennia marina

and abscisic acid, 324dehydration damage, 317desiccation tolerance, 312, 313germplasm distribution, 334maturity indicators, 320rehydration, 316storage longevity, 325

axescryopreservation, 328-333

thawing, 333

axes (continued)deterioration, 278and dormancy, 177equilibrium moisture, 56heat of sorption, 309and priming, 293and transportation, 322and water, 102, 309, 310of wild rice, 319

Azadirachta indica, 329

Baccaurea, 308baking, 206-207barley

crop management, 446-448cultivar choice, 447dormancy

genetics, 184, 213hull effect, 171, 173-175, 180,

204-205prediction of, 188release from, 182, 186and temperature, 180-181

genetics, 184, 213, 444maturity, 107and nitrogen, 435, 437, 438

fertilizer, 447-448oxygen, 105preharvest sprouting, 200quality

breeding for, 444-446environmental factors, 441-443,

448nutrients, 442structural factors, 429, 433-441

Spain versus Scotland, 436-439sulfur deficiency, 374temperature, 108, 180, 181uses, 208, 360. See also maltingwater, 181, 436, 441Web site, 445world trade, 352

barnyard grass, 110barometric pressure, 19, 211base excision repair pathway, 290base water potential

advancement below, 102and after-ripening, 63, 257

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base water potential (continued)and dormancy, 63, 247, 256-259germination below, 65-68and priming, 141, 147and seed maturity, 103and seed moisture, 65-68

batters, 207bedding plants, 138beets

germination control, 23mitosis, 148pelleting, 128priming, 134, 150vigor screening, 114

benzyl adenine, 138-1,3-glucanase, 147, 228-(1 4)mannan polysaccharides, 145

beta-glucan. See -glucan-glucan

in barley, 434breeding for, 443and drought, 442model, 436in oats, 361and temperature, 439, 440

-mannanase, 145-147, 227, 231-233-tubulin protein, 148, 149

biopriming, 138bluegrass, 102Borago officinalis, 400Brassica spp. See also rapeseed

and abscisic acid, 224-225antinutritional compounds, 410-411and glucosinolates, 411pelleting, 128phytosterols, 405seed vigor, 107

brassinosteroids, 235bread, 204, 209-210, 357-360, 380breeding

for malting, 444-446oil crops, 398, 403, 410preharvest sprouting, 212

brewing, 430-433broccoli, 103Bromus tectorum, 63, 256-259, 264buoyancy sorting, 126buried seeds. See also depth

and dormancy, 249, 254, 257

cabbage, 127red, 107

cacao, 322Calamus sp., 328calcium, 135, 206, 331caleosins, 396Camellia sinensis

cryopreservation, 329, 332desiccation sensitivity, 310embryonic axis, 309storage medium, 324

cancer, 406, 409canola. See also rapeseed

and dormancy, 170fatty acids, 399name, 411and oleic acid, 399phenolics, 410phytic acid content, 409phytosterols, 405polymeric coating, 129, 130-131protein content, 408

canopies, 252carbohydrates, 101, 151-153, 442

complex, 354carbon dioxide, 7, 19carbonyl groups, 154carboxypeptidases, 204carotenes, 404carotenoids, 404carrots

and aggregates, 23dehydration, 153embryos, 144and emergence, 80pelleting, 128priming, 134, 137, 147and temperature, 67, 69

caryopsesand dormancy, 171, 175-177, 187of wild rice, 319

Castanea sativa, 308, 315castor bean, 391, 399castor toxins, 412catalase

and cold, 113and dough, 204and free radicals, 154, 289and osmopriming, 294

cauliflower, 53, 149, 155

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celeryand aggregates, 23embryos, 144pelleting, 128priming, 138, 147sowing system, 131

celiac disease, 353cell cycle, 147-149, 155, 286, 313cell membranes

and desiccation, 152-154and deterioration, 283and fatty acids, 399integrity, 291and maturation drying, 313oxidation protection, 403and phytosterols, 405

cell wallsand embryo growth, 145-147, 224and endosperm, 227extensibility, 157, 224, 228, 234and genetics, 157and gibberellins, 236and malting, 430, 434oat endosperm, 361

cellscytoplasm, 152and desiccation tolerance, 152, 312and germination, 226mitochondria, 285-288, 313priming effect, 147

cerealscross section, 356fdormancy, 170-177, 180, 188-190,

204economic effect, 200fiber in, 353-355, 360, 361nutritional value, 353-355preharvest sprouting

causes, 199control of, 204-206economic effect, 200measurement, 209-212process, 201-204quality issues, 206-209

processing, 352-353stand establishment, 36

chelation, 206, 409Chenopodium album, 80, 251, 255, 263chickpeas, 26, 105, 112chicory, 128

chilling sensitivity, 105, 112, 130, 324chitinase, 147chitting, 25chloroform, 127chlorogenic acid, 410chlorophyll, 127cholesterol, 406, 409chromanols, 402-403chromosomes, 285, 444. See also

geneticscinnamic acid, 291Clark’s Cream wheat, 213Clausena lansium, 310clay domains, 9-10climate, 10-12, 21. See also rainfall;

seasons; temperatureclover

coating systems, 129critical water potential, 26kura, 101red, 99-100

coating techniques, 103, 128-131coconut oil, 396coconuts, 322, 331Coffea spp., 308, 309, 310, 332cold storage, 326-327cold tolerance, 105, 112, 130, 324cold unit sums (C), 254-255coleoptiles, 108, 110, 111colloids, 101color sorting, 127coloration, 410, 411colored seeds. See also pigments

manmade, 128, 129natural, 111, 127

complex carbohydrates, 354contact, seed-soil, 21, 32, 55, 77, 82containers, 324, 334corn. See also maize

critical water potential, 26H-SPAN use, 104herbicides, 112hypoxia, 104, 110pelleting, 128priming, 294, 295season length, 53seed harvesting, 107seed size, 100sowing, 109and temperature, 113

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cost effectiveness. See also yieldand malting quality, 443of oil crops, 412preharvest sprouting, 200and priming, 156

cotton, 26, 411cotyledons, 102, 278, 308. See also

dicots; monocotscowpeas, 104, 105, 112critical water potential, 26, 28crop density, 53crop management, 446-448cryostorage, 328-335

thawing, 333cucumbers, 127

cultivar differencesand crop management, 446dormancy, 171, 181temperature, 105water stress, 102

cytokinins, 138, 206cytoplasm, 152

databases, 35Datura ferox, 224, 226, 229-231day length. See diurnal variationsdehydration. See also desiccation

tolerance; recalcitrant seedsof excised axes, 331rate of, 153, 317, 330and reactive oxygen species, 314and temperature, 318tolerance comparison, 307-310

dehydrogenases, 294-295density

of plants, 53seed sorting by, 127

deoxyribonucleic acid (DNA)and barley breeding, 444mitochondrial versus nuclear, 286oxidative damage, 285-287and priming, 294repair, 154, 286, 316

mismatch repair pathway, 290replication, 313synthesis, 148, 280, 293

depthand aeration, 8, 19and dormancy, 250and heat transfer, 16

depth (continued)and pesticides, 111of sowing, 4, 77, 83, 108

variable, 109and water content, 12

desiccationdamage sensu stricto, 317, 326, 327,

332and dormancy, 173, 179, 190effects, 151-155

desiccation tolerance. See alsorecalcitrant seeds

induction of, 132, 149, 315LEA role, 315markers, 156mechanisms, 312-316and radicle growth, 56, 72and sugars, 151-152

deterioration. See also free radicalsdefined, 273effects of, 280-281factors, 275, 285glass formation, 314intraseed variation, 278, 279, 296modeling, 295, 296physiology, 278-280in recalcitrant seeds, 323-326repair, 292-296, 316and temperature, 275, 283

dew, 199dicots, 278, 306-307, 351differential display, 187differential scanning calorimetry, 153diffusivity, 27, 30-31, 55

soil-air, 19dikegulac-sodium, 291disease resistance, 111disinfecting, 137-138dispersion, irregular, 4disulfide bonds, 371, 375diurnal variations

and dormancy, 188and soil, 15, 16, 17and temperature, 250and water, 15

DNA. See deoxyribonucleic aciddormancy

and abscisic acid, 171-175, 178-181,186, 187, 190, 205, 235

benefits, 221

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dormancy (continued)buried seeds, 249, 254, 257in cereals, 170-177, 180, 188-190,

204cycling, 246defined, 169, 246degrees of, 247, 252and emergence, 54and endosperms, 170and environment, 187-191, 221,

248-250, 252genetics, 177, 183-188, 190, 213and malting, 169, 182molecular biology, 186-188and preharvest sprouting, 204-205primary versus secondary, 246,

254-256and recalcitrance, 274, 319release from

and gibberellens, 233-235light effect, 222-224population model, 259and seasons, 246and temperature, 250, 261-264and water potential, 257

residual, 71-72and seasons, 70, 246, 248, 250, 274and soil water content, 264in sunflowers, 170, 177-180, 181and temperature, 180, 183, 188-190,

247-250, 254, 263and water potential, 63, 247,

256-259and water uptake, 25in weeds, 246, 251, 264

modeling, 253-264dough

elasticity, 376quality, 204, 368-370, 374,

376-379prediction of, 377-379

stickiness, 207strength and viscosity, 351, 379water washing, 358

drift, 128drills, 127-128drought. See also water stress

tolerance of, 114, 137. See alsodesiccation tolerance

drum priming, 136

dryingafter priming, 138-139flash drying, 331maturation onset, 313partial, 327prestorage, 318slow versus fast, 317

Dumas analysis, 368Durio zibethinus, 318durum wheat, 377, 379dwarfing, 138

ecology, 155economics. See cost effectiveness; trade

statistics; yieldeggplants, 294, 295Ekebergia capensis, 312electrical conductivity, 130, 294electrolytes, 294electromagnetics, 139Elymus elymoides, 66, 69embryos. See also axes; dormancy

confining structures, 144cryopreservation, 334desiccation-tolerant, 312genetics, 157gibberellins, 233, 236growth potential, 157, 224-226, 236light, 236and malting, 430of oil crops, 391preharvest sprouting, 202priming, 144-149, 293types of, 144

emergenceand economic yield, 52-53and environment, 54H-SPAN effect, 104pesticide effects, 110and pigmented seeds, 111and plant size, 53prediction of, 72, 74, 80. See also

threshold modelsand priming, 133, 138promoters, 130of radicles, 145-149, 321and seed size, 99-101, 102simulations, 82-83and soil resistance, 82

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emergence (continued)and temperature, 56, 261-264timing of, 53, 56, 80and water-uptake rate, 55of weeds, 53, 54, 247, 255, 261

endive, 128endoplasmic reticulum, 153, 313endosperm plugs, 322endosperms

and aging, 293of barley, 430, 434, 437, 441of castor bean, 391cell-wall degradation, 145-147and dormancy, 170and light, 233, 236of maize, 365micropylar weakening, 226-233, 236of oats, 361and preharvest sprouting, 202, 204and radicles, 226of tomatoes, 144, 231and water potential, 144, 145-147of wheat, 370, 380

flour, 354-355energy

metabolism, 149-150. See alsometabolism

in milled wheat, 354radiant, 15-18

enhancement, 125. See also primingenvironment. See also light;

temperature; water potentialand barley, 441-443, 448and cultivar choice, 446and dormancy, 187-191, 221,

248-250, 252and emergence, 54for germination, 5-8modeling, 76-78and preharvest sprouting, 199and seed development, 106-108, 113and weeds, 264

enzymes. See also specific enzymesin animal feed, 409antioxidant, 153, 288-290, 294, 314,

403cell wall weakening, 157debranching, 203, 205and deterioration, 280, 283DNA cleaving, 155and fatty acids, 394-396

enzymes (continued)and heat stress, 440and malting, 430and oleic acid, 413and poisons, 412and preharvest sprouting, 202-205,

207-208and priming, 147, 294proteolytic, 203R-, 202and radicles, 145-147, 227and repair, 292, 297starch synthase, 381, 440and temperature, 18, 440and tomatoes, 145-147, 231

erucic acid, 399, 411etephon, 183ethylene

and cereals, 206and dormancy, 183and germination, 7, 183and mutations, 235priming with, 138and soil depth, 19and tillage, 110

ethylenediaminetetraacetic acid(EDTA), 206

Eucalyptus delegatensis, 78-80, 260Euphoria longan, 318, 332Euterpe edulis, 309evaporation, 23, 29, 201evening primrose, 400evolution, 274, 307expansin, 225, 234explants, 328-335expressed sequence tags (ESTs), 445

faba beans, 106Fabaceae, 98FAD2, 413falling number, 211far red (FR), 223, 225, 231, 251-252fatty acids

and breeding, 413-414essential, 399-400hypocholesterolemic, 399and lipid peroxidation, 282-285in maize, 367nutritional value, 399-401

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fatty acids (continued)in oils, 393-396and phytosterols, 405-406saturated, 396-398, 400, 413trans, 398in triacylglycerol molecule, 400-401unsaturated, 398-400

fermentation, 208, 430-432ferrous sulfate, 291fertilizers, 447-448fiber

and cell walls, 434and milling, 353-355in oats, 361in oilmeal, 406-407and seed part, 407in triticale, 360

Fick equations, 19field conditions, 78-83

deterioration in, 278and dormancy, 259, 265heat stress, 380prediction of, 37

filling, 309, 439-441, 442film coating, 129flash drying, 331flavinols, 152-153flax. See linseed oilflooding, 104, 110, 130flowering, 137, 446flowering plants

pelleting, 128priming, 134, 138, 153storage, 292

fluence. See low fluence responsefluorescein, 212fluorescence, 127fluridone, 171forestry, 126, 318-320Fourier’s law, 16free radicals

defenses against, 288-292description, 153-154in human beings, 402, 406and lipids, 282-283, 285mitochondria, 283-285, 297and moisture, 283-285and priming, 295and recalcitrance, 314, 316

French beans, 291, 294friabilin, 380

fructose, 154fungi

and deterioration, 278and in vitro germination, 330and late season, 310and priming, 294

fungicides, 110-112, 321, 326fungistats, 325

galactosyl cyclitols, 314gamma-linolenic acid, 400Garcinia gummi-gutta, 310Gaussian curves, 69gel electrophoresis, 361, 372, 373, 375gel encapsulation, 325gel phase, 315genetic engineering, 414, 445genetics. See also breeding;

quantitative trait lociand abscisic acid, 235and barley, 184, 213, 444and cryopreservation, 328, 330and desiccation tolerance, 312and dormancy, 177, 183-188, 190,

213for enzymes, 147, 157, 228-234and linseed oil, 400of maize, 366mutations, 285-287, 293and preharvest sprouting, 186, 187,

203, 212and sunflower oil, 413and temperature, 105-106of wheat, 372-373, 376, 380, 381

germination. See also priming;threshold models

and aeration, 7-8definition, 3, 140, 199and (de)hydration, 102and endosperm, 226-233and environment, 5-8inhibitors of, 205. See also abscisic

acid; lightin vitro, 330model variables, 29-36, 77nonagricultural, 32and oxygen pressure, 54and pelleting, 132percentage estimation, 259

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germination (continued)precocious, 171priming effect, 143, 146, 293promoters, 175-177, 182, 223, 228rate factors, 141, 143, 146soil factors, 11, 28

surface distance, 19, 73stimulation, 7, 18in storage, 309synchronization, 131-134and temperature, 71, 106, 180, 259.

See also temperature, andgermination

time to, 80-82, 132. See also timing,and germination

uniformity, 133, 141and water, 5-6, 24-26, 65-68

germplasm screening, 106gibberellic acid, 182, 183, 205gibberellins (GA)

deficiency, 233and dormancy, 175-177, 179, 233and embryo growth potential, 224and endosperm, 233-234and light, 224, 227, 234with priming, 138and seed coat, 236synthesis, 233-234

glassformation of, 291, 314stability, 292

glass transition temperature, 153gliadin proteins, 372-374, 377global positioning systems (GPS), 109,

110globulins, 150, 361, 363, 407glucose, 154glucosidases (maltase), 203glucosinolates, 410-411, 414glumellae

and dormancy, 173, 204-205germination constraint, 171and oil content, 412

glumes (bracts), 205glutathione (GSH), 289, 295glutathione peroxidase (GP), 289, 294glutathione reductase, 290-291glutelins, 363, 366gluten, 351, 358, 369-372glutenin, 371, 374, 375-380glycolase, 290

glyoxysomes, 295Golgi bodies, 313gossypol, 411-412G-protein-coupled receptors, 187grading, 126-127grains. See also cereals; filling; oil

crops; specific grainscoarse, 352. See also barley; oats;

sorghumhardness, 380processing, 353quality, 429, 433-441swathed, 214uses, 349-352, 355, 360

granule-bound starch synthase (GBSS),381

grasses, 36, 102, 110. See also ryegrasspriming, 134

groats, 361growth

down- and upward, 72-74of embryos, 145-149, 157, 224-226,

236impedance effect, 74, 84initiation, 56, 67and matric potential, 73models

preemergent seedlings, 74threshold, 84-85

predictions, 82regulation, 3, 128, 138slow growth storage, 328and soil surface, 82and sowing depth, 83and temperature, 73water potential effect, 73, 74

H-SPAN (hydrolyzed starch-graft-polyacrylonitrile), 104

Hagberg method, 211hardening, 137harvest, of seeds, 103, 107, 320heat capacity, volumetric, 16heat shock proteins, 153, 315heat of sorption, 309heat stress, 380, 440heat transfer, 16-17heat unit sums (H), 254-255herbicides, 53, 111-112

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Hevea brasiliensis, 327, 331, 332hexane, 127high irradiance response (HIR), 223,

231, 251high performance liquid

chromatography (HPLC), 443reverse-phase (RP-HPLC), 372

high red. See far redhordeins, 435, 442horticulture

coating systems, 129organic solvents, 127priming, 138recalcitrance, 318-320size grading, 53sowing systems, 127-128

hot water treatment, 137-138hull, 412. See also glumellaehumidification, 130, 293humidity, 199, 275, 285hydration. See also priming;

rehydrationand cell cycle, 149critical level, 26incomplete, 102and metabolism, 3, 102rehydration, 154as repair, 292-297, 297and water potential, 6

hydraulic conductivity, 14, 27, 55, 73hydrogen bonds, 151-153hydrogen peroxide, 114, 173, 282hydrogen sulfide, 183hydrolases, 227-228, 231

inhibitors of, 380hydrolysis

and nucleotide excision repair, 290and polysaccharides, 227and preharvest sprouting, 202-204,

212hydrolyzed starch-graft-polyacrylonitrile

(H-SPAN), 104hydrophilicity, 104, 131, 152, 275hydrophobicity, 130, 202hydropriming

description, 136and microorganisms, 138and proteins, 150, 157and UV absorption, 294

hydropriming time, 65-66, 68hydroseeding, 131

hydrothermal priming timeand cell cycle, 148in germination model, 142-143and hydrothermal time, 71, 78-82influencing factors, 66, 68

hydrothermal timedescription, 62-65and ecology, 155and hydrothermal priming time, 71,

78-82in model, 140-142

hydrotime, 60-61, 65hydroxyl radicals, 282hypocotules, 11hypocotyls, 82hypoxia. See also anoxia

and adventitious roots, 104in corn, 110and dormancy, 173, 180

imbibitionand ATP, 113chilling injury, 130and coatings, 129-131description, 14, 54-56and DNA, 148of dormant seeds, 187and free radicals, 295H-SPAN effect, 104lipid peroxidation, 285partial, 102and priming, 136and repair, 292-293and seed parts, 102and seed shape, 100and temperature, 201threshold models, 81

impatiens, 292impedance

and root penetration, 109seed-soil water flow, 33-35, 74

in vitro collection, 322in vitro germination, 330individual variation

axis water content, 310deterioration, 279dormancy, 248, 265minimum moisture, 102

indole-3-acetic acid (IAA), 206

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industrial uses. See usesinfrared spectroscopy, 149, 153Inga uruguensis, 324insecticides, 111-112interfaces, seed-soil, 21, 32, 55, 77, 82iron, 154, 291irrigation

after stress, 102and aggregate size, 23and emergence, 75and fungicides, 111and germination, 29and shoot elongation, 85

isocitrate lyase, 295

jasmonic acid, 206

Kjeldahl method, 368

labels, 322labor, 22Landolphia kirkii, 307, 310laser-induced fluorescence, 127late embryogenesis abundant proteins

(LEAs), 151-153, 155, 315leeks

maturity indicators, 127pelleting, 128priming, 134, 137, 147, 293

legumes, 99lentils, 71, 112, 291lettuce

abscisic acid, 235buoyancy sorting, 127embryo growth, 224emergence, 53endosperm, 144and gibberellens, 233and moisture minimum, 102pelleting, 128priming, 146repair, 292temperature, 147temperature and water, 63, 141and water potential, 63, 80

lightand carotenoids, 404and dormancy release, 222-224,

233, 251and embryo growth, 224-226and endosperm, 231, 234and priming, 294sensitivity to, 4, 250, 263and weeds, 251-252

light stress, 7linoleic acid, 399, 405, 413linseed oil

alpha-linolenic acid in, 399antioxidants, 403genetic engineering, 414toxins, 412

lipases, 204, 212lipid peroxidation

and cold tolerance, 113description, 282and mitochondria, 297and moisture, 283-285and priming, 294-295in storage, 294

lipidsdegradation of, 154in maize, 367in sorghum, 367and water uptake, 102

lipoxygenases, 283-285, 295L-isospartyl methyltransferase, 294Litchi chinensis, 310, 318low density lipoproteins (LDLs), 406low fluence response (LFR), 223, 225,

231, 251lumenal stress protein, 153Lupinus pilosus, 99lyophilization, 209lysine, 408

Machilus kusanoi, 311magnetic fields, 139maize. See also corn

and abscisic acid, 186amylose in, 366annual production, 352antioxidants, 405coating systems, 129cold-tolerant, 105

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maize (continued)deterioration, 276-278

pretreatment, 291emergence, 53endosperm, 365fatty acids in, 367genetics, 366glutelins, 366kernels, 365lipid components, 367pelleting, 128and phytosterols, 405precocious germination, 171preharvest sprouting, 200protein content, 366uses, 364-365water content, 56

malate synthase, 295malondialdehyde (MDA), 154maltase, 203, 205malting

definition, 430and dormancy, 169, 182and drought, 441-442and grain quality, 181, 429,

433-442breeding for, 444-446

and heat stress, 440-441quality analysis, 443sensitivity to water, 181yield, 434-435, 442, 447

maltodextrin, 152Mangifera indica, 318mannan, 227, 231mannitol, 135, 334mapping, 109-110markers

and barley breeding, 444desiccation tolerance, 156dormancy, 183-186, 257hardness, 380maturity, 107, 127, 320preharvest sprouting, 211

resistance to, 213priming, 149, 150, 154, 156

matric potential, 73, 278. See also soilmatric potential

matripriming, 294, 295matrix priming, 136, 138maturation drying, 313, 320

maturitydesiccation sensitivity, 318and dormancy, 188-190indicators, 107, 127, 320preharvest sprouting, 203residual chlorophyll, 127and temperature, 107uniformity at, 53and water, 103

mechanical impedance, 84-85melons

embryo expansion, 233priming, 142, 292, 295

membranesof cells. See cell membranesof mitochondria, 285, 288around oil bodies, 396of seeds, 27, 275, 283, 295, 296

mercuric chloride, 331meristem, 293mesocotyls, 101metabolism

in aged axes, 293and energy, 149-150of fatty acid hydroperoxides,

283-284and oxygen, 7, 150in recalcitrant seeds, 309-310, 321,

326and water, 12, 102, 149

hydrotime, 62, 65metabolism-derived damage, 317, 326,

327, 332metal ions, 154, 409microorganisms, 137, 138, 320microsomal oleate desaturase (FAD2),

413millet

deterioration, 291moisture stress, 104preharvest sprouting, 200temperature, 106uses, 367

millingbarley, for malt, 432rice, 363-364sorghum, 367wheat, 354, 356f, 359, 380

mitochondria, 285-288, 313mitosis, 147-149, 155, 286, 313

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modeling. See also threshold modelsafter-ripening, 256-259, 264deterioration/repair, 295-296dormancy, 188-190

release, 259, 261-264in weeds, 253-264

emergence patterns, 82, 84of environment, 76-78and field conditions, 37germination variables, 29-35of growth, 84

preemergent, 74hydrothermal priming time,

140-142hydrothermal time, 140-143imbibition, 81of light sensivity, 263limitations of, 77-78malting, 443-444predictive value of, 75-77preharvest sprouting, 189seedbed structures, 35-36seedling growth, 84-85weed emergence, 261

moistureabsorption of, 201in seeds, 56, 275, 283-285, 292in soils, 10, 249-250stress, and wheat, 113and temperature, 6

molds, 111, 208molecular biology, 186-188molecular diffusion, 19monoclonal antibodies, 212monocots

grains, 351, 356f. See also cerealsseeds, 72, 278, 307

monophenol oxidase, 204Morex, 184mulch, 18, 110-111mungbeans, 141mustard, 291, 399myxospermy, 103-104

neem, 329Nephelium lappaceum, 318nephelometry, 211-212nitrate, 247, 255, 264

nitrogenand barley

fermentability, 437fertilization, 447-448hordeins, 435and yield versus quality, 442

cryopreservation, 332-334oats, 362rice, 363roles of, 442and temperature, 438, 440and water uptake, 101

noodles, 204, 208, 358, 379Japanese, 381

nucleosome oligomers, 155nucleotide excision repair, 290

oatsannual world trade, 352cold tolerance, 113(de)hulled, 361dormancy, 186, 187endosperm, 361fat composition, 361industrial uses, 360-362preharvest sprouting, 200

Oenothera biennis, 400oil bodies, 396oil crops

breeding, 398, 403, 410, 411,412-414

human health factors, 396-398,401-406

listing, 390t. See also specific plantsoil content, 390t, 412profit optimization, 412protein content, 407-408quality

of grain, 391-392of oil, 392-406, 412-414of oilmeal, 406-412versus yield, 414

uses, 389-391, 399, 400oilcake. See oilmealoilmeal

genetic engineering, 414quality, 391, 406-412of rapeseed, 392toasting, 411uses, 389, 406

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oleic acid, 399, 405, 413oleosins, 396oligosaccharides, 151-153, 291-292,

314-315olive oil, 399, 405, 406onions

burial depth, 82-83crop density, 53emergence timing, 80hydrothermal time, 141pelleting, 128, 130priming, 134, 135-136, 147, 294and temperature, 67, 69

organic solvents, 127ornamentals, 127, 134. See also

horticultureOrobanche aegyptiaca, 69orthodox seeds

continuum, 311description, 273-275, 305deterioration, 280-288maturation drying, 313

Osborn procedure, 363, 366osmopriming

aeration, 135, 150and antioxidants, 154and -mannanase, 146with biopriming, 138and cell cycle, 142in corn, 294and DNA repair, 154-155and lettuce, 293and microorganisms, 173in muskmelons, 292in orthodox seeds, 316and proteins, 157and RNA, 293and storage proteins, 150and temperature, 142

osmosis, 12osmotic stress, and germination time,

113osmotic water potential, 28-29, 224, 226

virtual (VOP), 67, 78, 143overgrazing, 4oversowing, 53oxalate oxidase, 212oxidative damage, 153-154. See also

free radicalsoxidative phosphorylation, 285-288

oxygenactive oxygen species (AOS),

153-154. See also freeradicals; reactive oxygenspecies

and cold tolerance, 113critical concentration, 105and desiccation, 149and emergence, 54and fatty acids, 398, 399and germination, 7lack of. See anoxia; hypoxiaand light, 7singlet, 282and soil, 15, 19, 110and temperature, 7, 15and water, 7, 13, 19

oxygen diffusion coefficient, 19-20oxygen partial pressure, 54

packaging, 321-322packing media, 323-324paclobutrazol, 138, 177, 234palm oil, 396, 401

antioxidants in, 403, 404pasta, 204, 208, 358, 379peanut oil, 408, 410peas, 291, 293, 295pelleting, 128, 132penetrometer pressure, 84-85pentosans, 434peppers

embryos, 148endosperm, 144maturity indicators, 127pelleting, 128priming, 134, 148, 153, 155, 294storage longevity, 292

peptidases, 203pericarp

dormancy, 170, 179, 204fungi, 326preharvest sprouting, 202

perisperm, 233permeability

and polymers, 130and priming, 135-136rapid versus delayed, 98of seed coats, 31-33, 55, 98-99

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peroxidase, 204, 295pest resistance, 111pesticides, 110-112, 129petroselinic acid, 399phenolics, 99, 405, 410phospholipids, 396phosphorus, 204, 409-410phosphorylation, 187photoacoustics, 149photoelectric cells, 127photooxidation, 404photoreceptors, 222-224photosynthesis, 441phytases, 204, 205phytates, 409-410phytic acid, 206, 409-410phytochromes

and cell wall, 224, 234and embryo growth, 236forms and action, 251and germination, 223, 224model, 263

phytosterols, 405-406pigeonpeas, 290pigments. See also phytochromes

carotenoids, 404chlorophyll, 127pest resistance, 111and preharvest sprouting, 205, 213

plasmalemma, 313Podocarpus spp., 308polyacrylamide, 131polyethyleneglycol (PEG), 135, 293,

324polygalacturonase, 147Polygonum aviculare, 249, 250,

255-256, 259Polygonum persicaria, 254, 255, 263polymerase chain reaction (PCR), 287polymeric polyphenols, 4, 367. See also

tanninspolymers

coatings, 103, 129-131priming, 136and soil matric potential, 104

polyphenol oxidase, 173, 204, 208polysaccharides, 145, 227, 433-434pore exclusion principle, 13porosity, 10, 99, 146potassium, 135, 224, 334pregermination, 131-132

preharvest sprouting. See also specificgrains

causes, 199control, 204-206, 212-214definition, 199and dormancy, 204economic loss, 199-201enzymes, 202-204genetics, 186, 187, 203, 212measurement, 209-212quality issues, 206-209resistance model, 189sampling, 209

priming. See also hydrationand active oxygen species, 153-154and base water potential, 65-67basic premise, 25and buoyancy sorting, 126and cell cycle, 148-149cost effectiveness, 156description, 132-134(dis)advantages, 131-133and DNA, 154-155drying after, 138-139, 146and ecology, 155and embryos, 144-149and enzymes, 147, 294and germination, 143, 146, 293and hydropriming time, 65-67hydrothermal models, 140-143markers, 149, 150, 156methods, 134-139

optimal, 134-135microorganisms, 137, 138natural, 102overpriming, 149, 151, 155and permeability, 135-136and proteins, 150and radicles, 148and repair, 292-296and temperature, 136, 141-143, 148time factors, 148, 293of tomatoes. See tomatoes, priming

probit analysis, 59-60, 62, 63prolamins, 361, 363protein kinases, 187proteinases, 204, 205, 208, 212protein(s). See also late embryogenesis

abundant proteins; storageproteins

-tubulin, 148, 149and desiccation, 151

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proteins (continued)and deterioration, 280and dormancy, 179endosperm-weakening, 234heat shock, 153, 315heat-soluble, 155lumenal stress, 153in maize, 365-366and malting, 434, 442, 447in oats and rice, 361-363in oil bodies, 396in oilmeal, 389, 391, 407of oilseeds, 407-408preharvest sprouting, 207and priming, 150solubility, 407-408in sorghum, 367stress, 153VP, 179and water uptake, 101-102in wheat, 206, 368-380

Pseudomonas, 138puroindolines, 380Pythium ultimum, 138

Q10 factor, 6, 18defined, 15

qualityassessment of, 107, 443of barley

analytic methods, 443environmental factors, 437-442nutrients, 442structural factors, 429, 433-441

in oil crops, 391-406, 412-414and preharvest sprouting, 206-209and recalcitrant seed storage,

324-325sorting and grading, 126-127and sprouting

finished products, 206-209seeds, 208-209

versus yield, 414, 442of wheat, 368-381

quantitative trait loci (QTL)and malting, 184, 213, 444preharvest sprouting, 187and sorghum, 186

quarantine, 322Quercus alba, 307-308, 320

Quercus faginea, 331Quercus negra, 307-308Quercus robur

cryostorage, 331, 333dehydration, 308desiccation tolerance, 310storage, 309, 323, 326

R-enzyme, 202radicles. See also roots

and desiccation, 56deterioration, 276-278

repair site, 293development factors, 104, 132and DNA synthesis, 148and embryo growth, 224emergence, 145-149, 321and endosperm, 226enzymes, 145-147, 227growth

precondition, 67rate of, 78, 80

and hypoxia, 110and overpriming, 151and oxygen, 7and soil, 11and water, 6, 25, 102

radishes, 294, 295raffinose oligosaccharides, 151-153,

291, 314rainfall

after stress, 102and dormancy, 189and mitosis, 148and preharvest sprouting, 199

random amplified polymorphic DNA(RAPDs), 444

rapeseed. See also canolaantinutritional content, 411, 414breeding advances, 391and environment, 106, 107genetic engineering, 414oilmeal, 392oils from, 399phenolics, 410phytic acid content, 409and phytosterols, 405protein content, 408

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reactive oxygen species (ROS),287-288, 314, 317, 326

recalcitrant seedscells, 312-316damage repair, 316description, 273-275, 305-311glass formation, 315and harvest, 320and LEAs, 315metabolism, 309-310nontropical examples, 307, 329prestorage drying, 318and seasons, 307, 310-311size of, 330species variation, 307-308storage, 323-328

cryostorage, 328-335transportation, 321-323

red clover, 99-100red wheat, 201, 205, 213regression analysis, 59, 62, 63rehydration

amphipathic substances, 315damage repair, 316dormancy, 179, 250free radicals, 154

repairof DNA, 154, 286, 290, 316of seed damage

location, 293mechanism, 294modeling, 296orthodox versus recalcitrant

seeds, 316timing, 292-293

respirationand deterioration, 275, 285-288,

292, 297of fungi, 321H-SPAN effect, 104stages, 7water and temperature, 82

restriction fragment lengthpolymorphism (RFLP), 183

retention curve, 13ribonuclease, 205ribonucleic acid (RNA), 293-294

mRNA, 186-187, 206, 445rice

brown, 363dormancy, 171, 186, 205, 213

rice (continued)industrial uses, 363-364lipoxygenase-deficient, 284North American wild, 319and oxygen, 110preharvest sprouting, 200pretreatments, 291QTLs, 213steeping, 137world production, 351

ricin, 412ricinoleic acid, 399Ricinus communis, 412ridges, 23, 36RNA. See ribonucleic acidroots. See also radicles

adventitious, 104-105penetration resistance, 109and water, 6, 73

ROS. See reactive oxygen speciesRumex spp., 254rye

accelerated aging, 155hybrid. See triticaleindustrial uses, 359-360preharvest sprouting, 200sulfur deficiency, 374world production, 351

ryegrass, 101, 102, 104

safflower, 398, 399, 400, 407saline conditions, 34Scadoxus membranaceus, 307screening protocols, 106, 112-114scutellum, 202-203seasons

and barley, 435and dehydration, 309and dormancy, 70, 248, 250, 274

exit from, 246and emergence, 53and germination, 70and recalcitrance, 307, 310seedbed preparation, 22and soil, 16, 17-18

seed coatsof cereals, 201, 205damaged or thin, 130and deterioration, 278diffusivity, 32

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seed coats (continued)and dormancy, 177, 179-181, 204and embryo growth, 224-226enhancement of, 103, 128-131and gibberellins, 236and hydration, 278and imbibition, 130light effects, 222-224and oxygen, 7permeability, 31-33, 55, 98pigment, 111, 127preharvest sprouting, 205and water potential gradient, 27and water stress, 98-99and water transport, 98weight, 99

seed furrow amendments, 103-104seed lots, 140-141, 142, 156seed membranes, 275, 283, 295,

296-297seed tapes, 131, 132seedbeds

aggregates, 23defined, 20and emergence, 53-56, 54indices, 22-23modeling, 35-36, 76-78ridged, 23, 36tillage, 20-22and water flow, 33-34

seedlings. See also emergenceflush prediction, 80pesticide uptake, 111-112postgermination growth, 72-74preemergent growth, 74

seeds. See also orthodox seeds;recalcitrant seeds

age, 105development, 106-108, 113, 150earliest, 274equilibrium moisture, 56harvest of, 103, 107, 320mucilagenous, 7, 103shape, 100, 127, 128size. See size, of seedssoil interface, 32-35synthetic, 334-335types, 273-275, 311variation, 70, 248-249water potential, 5, 27

seeds (continued)water requirements, 6water transport within, 30water uptake, 30-34, 100-104, 140

semiarid conditions, 8sequence, 3sesame oil, 405shattering, 107, 200shelf life, 132

of primed seeds, 133, 139shoots

apices, 334elongation, 84-85weight, 101

Shorea sp., 328signaling, 187, 234-235, 237f, 405sinapine, 410singlet oxygen, 282Sisymbrium officinale, 250, 255size

of aggregates, 21of cells, 147grading by, 53of seeds

and emergence, 99-101, 102enhancement, 128recalcitrant seeds, 330and soil aggregates, 23and sowing depth, 108and water uptake, 100weight, 74, 82

of starch granules, 381, 433, 440slaking, 10-11, 13slurry, 131-132sodium salts, 135, 331soil capillary water conductivity, 14soil characteristic, 13soil crust

potential for, 109, 111and radicle length, 104and seed size, 101

soil matric potential, 12-14, 28, 73, 104soil strength, 8, 54, 73-74soil surface, 19, 73, 82soil water characteristic hysteresis, 13soil water content

and after-ripening, 264soil-air diffusion, 19soils

aeration regime, 18-20aggregate size, 21, 23

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soils (continued)in arid zones, 4and climate, 11compaction

benefits, 8effects, 10, 29mapping, 109and matric potential, 13oxygen supply, 20remediation, 11

critical thresholds, 109diffusivity to water, 30-31disease-infested, 294fast-wetting effect, 13and germination, 8layered, 18mapping, 109-110modeling, 35-36moisture, 249-250nitrogen content, 447-448penetration resistance, 82and pesticides, 111-112porosity, 12-13seed interface, 32-35, 82shrinking, 29solid phase, 9-11stratification, 255, 259, 264sulfur-deficient, 373-374temperature

aeration, 19dependencies, 6and dormancy, 248, 254-256,

261-264heat transfer, 16-17and seed requirements, 7thermal capacity, 15-18volumetric heat, 16

tillage effect, 21water conductivity, 14, 27, 55, 73water content

diurnal variations, 15and dormancy, 264measurement, 77storage capacity, 109volumetric, 11, 16, 19, 29

water flow, 5, 14, 33-35, 74water potential, 12, 27

soluble starch synthase, 440sorghum

critical water potential, 26and diurnal temperature, 251

sorghum (continued)dormancy, 175-177, 180, 183, 186

release from, 251names, 367nutritional value, 367pigmented seeds, 111preharvest sprouting, 200and seed moisture, 103and soil crust, 101, 104tannins, 367-368trade statistics, 352uses, 367

Sorghum halepense, 261sorption, heat of, 309sorting, 126-127sowing

aerial, 128hydroseeding, 131precision systems, 127-128window for, 130-131

soybeansantinutritional content, 411deterioration, 278

protection steps, 290, 291and dormancy, 170fatty acids, 398, 399, 401genetic engineering, 414oilmeal, 389, 407, 408and oleic acid, 399phosphorus, 410porosity, 99priming, 294stearic acid, 398temperature, 105, 112vitamin E, 290water uptake, 55, 56, 98-99,

101-102yield, 412

species differencesrecalcitrance, 307, 317temperature, 6, 263

Spergula arvensis, 255, 263spring wheat, 100, 113sprouting, 170, 181, 204, 211. See also

preharvest sproutingsqualene, 406stachyose oligosaccharides, 314-315stand establishment

marginal conditions, 4, 21and pesticides, 110

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stand establishment (continued)remediation, 36soil factors, 11

stanols, 406starch. See also carbohydrates

barley, 433, 438, 440, 441granule-size, 381, 433, 440maize, 366sorghum, 367wheat, 358, 380-381

starch synthasegranule-bound, 381soluble, 440

stearic acid, 398steeping, 136-137Steptoe/Morex (S/M), 184sterilization, 322, 325-326, 330-331stirring number, 211storage

cryostorage, 328-335and dessication tolerance, 151-155and deterioration, 275, 295, 314

pretreatments, 291dry, and dormancy, 173-180, 179,

249for four years, 294germination in, 309and lipases, 204lipid peroxidation, 285, 294longevity factors, 291, 324-325of North American wild rice, 319of oil crops, 399, 401-402of recalcitrant seeds, 323-335

packing media, 323-324short- to medium-term, 323-328versus orthodox seeds, 273-275,

309of seeds, 132

and priming, 133, 139, 151-155slow growth, 328subimbibed, 327temperature, 275-276, 324-325

stratification, 255-256, 259-260time in, 63of water, by soil, 109

storage proteinsin barley, 438in barley, and malting, 434-437in oilseeds, 407-408and priming, 150

storage proteins (continued)and temperature, 440and wheat quality, 369-374and yield, 434-435, 442, 447

storage tissue. See also cotyledonsmoisture in, 56of oil crops, 391proteins. See storage proteinsreserve exhaustion, 74starch and lipid, 7

stratification, 255, 259, 264stratification thermal time (Stt), 260stress

and genetics, 157and germination, 29, 102moisture, 104. See also water stressand priming, 133screening, 106, 112-114from temperature, 113-114, 380, 440

stress proteins, 153Stt (stratification thermal time), 260stubble-mulch tillage, 110subimbibed storage, 327sucrose, 151, 291, 314sugars

cell stabilization, 151and deterioration, 285, 291and priming, 294in recalcitrant seeds, 314

sulfur, 373-374, 408sunflowers

coating systems, 129deterioration protection, 291dormancy, 170, 177-181, 190

release, 183fatty acids, 398, 401fiber, 407genetic engineering, 414and glutathione, 291oilmeal color, 410oleic acid, 399, 414pelleting, 128priming, 154, 294proteins, 408stearic acid, 398temperature, 413tocopherol, 403

superoxide anions, 282superoxide dismutase (SOD), 154, 289,

290, 294

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superoxide radicals, 153-154. See alsofree radicals

surfactants, 131swathed grain, 214swelling, 29synthetic seeds, 334-335

tannins, 367, 405, 410Telfairia occidentalis, 319temperature

and after-ripening, 256-259and barley, 437-440cold tolerance, 105, 324and coleoptiles, 108constant versus fluctuating, 18, 71,

106, 361-264and dehydration, 318and deterioration, 275, 283and dormancy, 180, 183, 188-190,

247-250, 254, 263release from, 250, 261-264

and emergence, 56of weeds, 261-264

and enzymes, 18, 440during filling, 309, 439-440fluctuations, 71, 106, 261-264and genetics, 105-106and germination

mean lower limit, 259percentage, 69rate of, 71, 141threshold models, 57-60, 62-65,

66-72, 259glass transition, 153heat sum approach, 74-75heat transfer, 16-17and hydraulic conductivity, 55and imbibition, 201and malting, 434and maturity, 107and nitrogen, 438, 440and oil crops, 413optimal range, 69, 72, 113, 140

sub- and supra-, 57-60, 63,68-70, 112, 140

and polymeric coatings, 130and preemergent growth, 73and preharvest sprouting, 199and priming, 136, 141-143, 148

temperature (continued)and proteins, 440and seed development, 113and seed storage, 132and soil

aeration, 19dependencies, 6dormancy, 248, 254-256,

261-264seed requirements, 7thermal capacity, 15-18volumetric heat, 16

species differences, 6, 263and storage, 276, 324, 326

cryostorage, 328-335stress screening, 112-113thermal window, 140and water potential. See water

potential, and temperatureterbufos, 111-112testa. See seed coatsthermal amplitude, 251thermal conductivity, 16thermal range, 254-256thermal time

and dormancy, 248, 257-259, 264,265

equation, 58and germination rate, 141seedling growth, 84-85uses, 74

thermal window, 1403- -hydroxylase, 233threshold models

dormancy, 259-260, 265seedling growth, 84-85temperature, 56-60, 62-65, 68-72virtual osmotic potential, 67-68water potential, 60-68

tillagefor conservation, 110seedbed preparation, 20-22, 36and soil structure, 10

timingof dormancy, 169-173, 177, 179, 188

exit from, 183, 190and emergence, 53, 56, 80of flowering, 137and fungi, 310and fungi attack, 294

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timing (continued)and germination, 29, 56, 80-82of growth initiation, 56of heat stress, 440and herbicides, 53to maturity, 53of nitrogen availability, 442of pelleting, 132of planting, 109of priming, 148, 293of repair, 292-293of seed harvest, 103, 107and temperature. See hydrothermal

priming time; hydrothermaltime; thermal time

and threshold models, 75-77of water stress, 442

tobacco, 147, 228tocopherol, 290, 291, 402tocotrienols, 402-403tomatoes

buoyancy sorting, 127cell separation, 227-228cell wall expansion, 225cell wall weakening, 157cold-tolerance, 105embryos, 148endosperm, 144, 231enzymes, 145-147, 231gibberellen deficiency, 233hydrothermal time model, 141maturity indicators, 127oxygen, 105pelleting, 128photoreceptors, 223priming

ATP:ADP, 150-mannanase, 146

cell cycles, 142, 148germination uniformity, 134and L-isospartyl

methyltransferase, 294postpriming heat, 153potassium salts, 135triazoles, 138

root tip abberation, 293sowing system, 131storage longevity, 291and temperature, 68water needs, 66, 102

total water potential, 5, 27toxic compounds, 408-412, 414trade statistics, 352transportation, 321-323trays, 128treatments. See also priming

antifungal, 110-112, 321, 325-326and deterioration

as cause, 279, 330as protection, 291

with hot water, 137-138sterilization, 322, 325, 330types, 125-126

trees, 307-308, 320. See alsorecalcitrant seeds; specificspecies

triacylglycerols, 392, 396, 400triazole, 138Trichilia dregeana, 324, 326, 327triglycerides, 409triterpenes, 405triticale, 200, 360tritordeum, 360trypsin inhibitors, 411tryptophan, 206turgor, 67, 102, 145, 147

uniformityof deterioration, 276-278, 296of germination, 133, 141of plants, 53, 127, 138

usesbarley, 360oats, 360-362rice, 362-364rye and triticale, 359-360wheat, 357-359

vacuum-feeding, 128vapor, 55vapor phase, 81vernolic acid, 399very low fluence response (VLFR),

223, 233, 251vigor

and dormancy, 71-72indicators of, 100, 108

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vigor (continued)screening for, 114of seedlot, 106-108versus quality, 99-100

virtual osmotic potential (VOP), 67, 78,143

viscosity, 211vitamin A, 404vitamin C, 290vitamin E, 290, 291, 403vivipary, 200volumetric soil water content, 11, 16,

19, 29Vp1 gene, 186VP (protein), 179

Warburgia spp., 319, 329, 331water

and dormancy, 181, 264in excised axes, 331forces on, 12hot water disinfecting, 137-138intraseed transport, 30, 278loss of, 81, 320. See also desiccation

tolerance)and oxygen, 7, 13, 19and preharvest sprouting, 201-202in soil, 11-15, 23, 109, 264soil to seed flow, 5, 21, 27, 33-35

water flotation, 126water potential. See also base water

potentialand cell walls, 225critical, 26, 28definition, 12and emergence, 56and endosperm, 229-231measurement, 77osmotic, 28, 224, 226. See also

virtual osmotic potentialand phytochromes, 224and priming, 155and seedlings, 73

preemergent, 6, 74, 113of seeds, 27-29in shoot elongation model, 85and sowing depth, 83

water potential (continued)and temperature, 62-69, 71, 85, 113.

See also hydrothermalpriming time; hydrothermaltime

total, 5, 27water potential gradient, 5-6, 14, 27water replacement hypothesis, 152water stress

cultivar difference, 102and dormancy, 188and dry soil surface, 82flooding, 104, 110, 130and germination time, 113and malting, 441-442and metabolism-linked damage,

317, 326and oxygen, 7and seed development, 107and seedling growth, 82, 84

water uptakein barley, 436and phenolics, 99by seeds, 30-34, 101-104

priming, 140size and shape, 100

Web sites, 445weeds. See also herbicides

dormancy, 246, 251modeling, 253-264release, 247, 251, 261-264

emergence, 53, 247, 255, 261germination, 70, 259and light, 251and seed size, 100and seedling flush, 80and sowing systems, 131temperature, 70, 113, 259water stress, 113-114

weightof seed coats, 99of seeds, 74, 82

wet conditions, 130. See also floodingwheat. See also winter wheat

Clark’s Cream, 213deterioration, 276

repair, 292, 293dormancy

QTLs, 183, 213release from, 186type of, 205

Page 501: Handbook of Seed Physiology Applications to Agriculture

wheat (continued)genetics, 372, 376, 380, 381

transgenic, 187gluten, 351, 358hybrids, 200, 360imbibition, 201intolerance of, 353milling, 354, 356f, 359preharvest sprouting, 199, 203, 205,

208-214resistance, 187, 205, 213

priming, 293, 294processing, 352-353quality factors

dough, 368-370, 374, 376-379hardness, 380product traits, 206-208proteins, 369-380seeds, 208-209starch, 380-381

red, 201, 205, 213seed size, 100, 101and soil aggregates, 23spring varieties, 110, 113and sulfur, 373-374and temperature, 105uses, 357-359variety identification, 372world production, 351

Wigandia urens, 155wind, 19winter wheat

and dormancy, 181and fungicides, 111seed size, 100, 102sowing depth, 108

xanthophylls, 404X-radiography, 127, 146xyloglucan, 227-228

yieldand crop density, 52-53factors, 446of malting, 434, 442, 447and pigmented seeds, 111and preharvest sprouting, 199-201and priming, 133, 137and seed size, 101versus quality, 414, 442

Zizania palustris, 319


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